Methods for plant transformation using spectinomycin selection

ABSTRACT

The present invention relates to methods and compositions for transforming soybean, corn, cotton, or canola explants using spectinomycin as a selective agent for transformation of the explants. The method may further comprise treatment of the explants with cytokinin during the transformation and regeneration process.

This application claims the priority of U.S. Provisional applicationSer. Nos. 60/894,096, filed Mar. 9, 2007, and 60/915,066, filed Apr. 30,2007, the entire disclosures of which are incorporated herein byreference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates generally to methods for preparing andtransforming meristematic plant tissue and selection and subsequentregeneration of transgenic plants.

2. Description of Related Art

Transformed plants may be obtained by directly treating meristematictissue of a plant embryo. The meristematic tissue contains formativeplant cells that differentiate to produce multiple plant structuresincluding stem, roots, leaves, germ line tissue, and seeds. Themeristematic tissue, such as soybean tissue, may be excised from seeds.Methods of genetically transforming soybeans (Glycine max) usingbacterially-mediated gene transfer directly on the meristematic cells ofsoybean embryos are known. Isolated cotton meristems and shoot apextissues have been transformed. Use of a cytokinin to induce shootdevelopment in tissue culture has been reported.

A number of selective agents are known for use in methods forgenetically transforming plant cells. Anaminoglycoside-3′-adenyltransferase has been used as a selectable markerin transforming plant cells. Fusion of aadA with a chloroplast transitpeptide-encoding sequence, to allow for directing a nuclear producedAadA to the chloroplast, has not been reported.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a method for producing atransgenic plant containing at least two heterologous nucleic acidsequences comprising: (a) providing an explant comprising a firstheterologous nucleic acid sequence that confers resistance to aherbicide; (b) transforming the explant to comprise a secondheterologous nucleic acid sequence comprising a selectable marker geneconferring spectinomycin resistance; and (c) regenerating an explantthat exhibits spectinomycin resistance into a transgenic plantcontaining at least two heterologous nucleic acid sequences. In oneembodiment, the explant comprises an embryonic meristem. In anotherembodiment, the first heterologous nucleic acid sequence confersresistance to glyphosate, bialaphos, phosphinothricin, Basta,glufosinate, 2,4-D, kanamycin and related aminoglycosides, hygromycin,an acetyl-coA carboxylase inhibitor, an oxygen radical generator, ordicamba. In another embodiment, the explant is a soybean, corn, cotton,or canola explant. In a particular embodiment, the explant is a soybeanor cotton explant, such as a soybean plant.

In another aspect, the invention provides a method of producing atransgenic plant comprising: (a) transforming at least a first seedexplant with a heterologous nucleic acid sequence comprising aselectable marker conferring tolerance to spectinomycin; and (b)regenerating a transgenic plant from the transformed cells, wherein theexplant is contacted, prior to, concurrently with, and/or following step(a) or step (b), with at least a first media comprising spectinomycin toselect transformed cells comprising said selectable marker. In oneembodiment, the transgenic plant arises from transformation of ameristem that results in transformation of germline tissue. In certainembodiments, the resulting plant is non-chimeric. In yet otherembodiments, the resulting plant is chimeric. In a particularembodiment, at least one shoot of the resulting plant is transgenic andis non-chimeric. In another particular embodiment, at least one shoot ofthe resulting plant is transgenic and non-chimeric while at least oneother shoot or one other root does not comprise a sequence comprised onthe heterologous nucleic acid. In certain embodiments the first seedexplant comprises a transgene. In other embodiments, the explantcomprises an embryonic meristem. In yet other embodiments, during orfollowing step (a), explants are grown in the presence of a selectiveagent at 35° C.-40° C. and/or are grown under lighting conditions thatallow for normal plastid development. In still yet other embodiments,growth at 35°-40° C. is performed for 1-7 days or the lightingconditions comprise at least 5 μEinsteins with about a 16 hour light/8dark photoperiod.

In some embodiments, the explant is stored at a temperature of between0-15° C. for between 1 hour and 7 days prior to step (a). In otherembodiments, the media comprises from about 15 mg/L to about 1500 mg/Lspectinomycin. The invention further relates to a method wherein thecells of the explant comprise a coding sequence conferring tolerance toglyphosate, bialaphos, phosphinothricin, Basta, glufosinate, 2,4-D,kanamycin and related aminoglycosides, hygromycin, streptomycin,ampicillin, or dicamba. In some embodiments, step (a) comprises growingan explant on a co-culture medium comprising spectinomycin. In otherembodiments the explant is not contacted with a medium comprisingspectinomycin after being transferred from a co-culture medium.Alternatively, in other embodiments the explant is contacted with amedium comprising spectinomycin after being transferred from aco-culture medium. In some embodiments the explant that is regeneratinginto a plant is transferred to soil or soil substitute for rootingwithout pre-rooting in aseptic media. In other embodiments, theheterologous nucleic acid further comprises a coding sequence thatconfers a trait of agronomic interest or improved end use.

In other embodiments, the invention provides a method of producing atransgenic plant comprising: (a) transforming at least a first seedexplant with a heterologous nucleic acid sequence comprising aselectable marker conferring tolerance to spectinomycin; and (b)regenerating a transgenic plant from the transformed cells, wherein theexplant is contacted, prior to, concurrently with, and/or following step(a) or step (b), with at least a first media comprising spectinomycin toselect transformed cells comprising said selectable marker, wherein step(a) comprises transforming the cell of the explant with at least asecond heterologous nucleic acid. In particular embodiments, the secondheterologous nucleic acid comprises a coding sequence that confersherbicide tolerance. In certain embodiments, the first and secondheterologous nucleic acids are integrated at different loci within thegenome of the cell. Certain embodiments of the invention comprise, priorto step (a), the step of priming the seed, wherein the priming comprisescontacting the seed with a cytokinin. In other embodiments a methodfurther comprising contacting the explant with a cytokinin prior to,concurrently with and/or following step (b) is contemplated. Inparticular embodiments, the cytokinin is selected from the groupconsisting of thidiazuron, BAP (6-Benzylaminopurine), kinetin, CPPU(N-(2-Chloro-4-pyridyl)-N′-phenylurea), 2iP (6-(y,y-Dimethylallylamino)purine), Zeatin, Zeatin-riboside, Adenine, and TIBA(2,3,5-Triiodobenzoic acid).

In some embodiments, step (a) comprises contacting the explant withrecombinant Rhizobiaceae comprising said heterologous nucleic acid,wherein the Rhizobiaceae have been exposed to thidiazuron prior to orconcurrently with contacting the explant with the recombinantRhizobiaceae. In certain embodiments the Rhizobiaceae is exposed tothidiazuron for from about 1 to 5 days prior to contacting the explantwith the recombinant Rhizobiaceae. In other embodiments, theRhizobiaceae are suspended in the presence of a selective agent activeagainst an untransformed explant prior to contacting the explants withthe Rhizobiaceae. In certain embodiments, the Rhizobiaceae are selectedfrom the group consisting of: Agrobacteria, Sinorhizobia, Mesorhizobia,and Rhizobia. In yet other embodiments, the explants are grown in thepresence of a fungicide prior to, during, or subsequent to the step oftransforming at least a first seed explant with a heterologous nucleicacid sequence comprising a selectable marker conferring tolerance tospectinomycin. In certain embodiments, the explants are grown in thepresence of a fungicide and DMSO. In particular embodiments, theexplants are grown in the presence of nystatin, thiabendazole, and DMSO.

In certain embodiments the explant is a soybean, corn, cotton, or canolaexplant. In particular embodiments the explant is a soybean explant or acotton explant.

In certain embodiments the method of: producing a transgenic plantcomprising: (a) transforming at least a first seed explant with aheterologous nucleic acid sequence comprising a selectable markerconferring tolerance to spectinomycin; and (b) regenerating a transgenicplant from the transformed cells, wherein the explant is contacted,prior to, concurrently with, and/or following step (a) or step (b), withat least a first media comprising spectinomycin to select transformedcells comprising said selectable marker, further comprises the step of(c) obtaining a progeny plant of any generation of the transgenic plantthat comprises the gene conferring the trait of interest and lacks theselectable marker. In certain embodiments, the heterologous nucleic acidcomprises a first DNA segment comprising left and right T-DNA bordersflanking a gene conferring a trait of interest; and a second DNA segmentcomprising a second set of left and right T-DNA borders flanking saidselectable marker conferring tolerance to spectinomycin. In otherembodiments, the method further comprises the step of (c) obtaining aprogeny plant of any generation of the transgenic plant that comprisesthe gene conferring the trait of interest and lacks the selectablemarker.

In some embodiments, the heterologous nucleic acid comprises right andleft T-DNA borders and first and second DNA segments, wherein the firstDNA segment comprises a gene of interest located after the right border,and wherein the second DNA segment comprises the selectable markerlocated after the left border. In certain embodiments, the heterologousnucleic acid comprises first and second right T-DNA borders, wherein afirst DNA segment comprising a gene of interest is located after thefirst right border and a second DNA segment comprising the selectablemarker is located after the second right border.

In certain embodiments, the method comprises culturing said explant onmedia lacking spectinomycin for from about 1 to about 7 days during step(b). In other embodiments, the method comprises contacting the explantwith at least a first media comprising spectinomycin is for from about15 minutes to about 7 days. In particular embodiments, the selectablemarker is encoded by aadA. In more particular embodiments, aadAcomprises SEQ ID NO:1. In certain embodiments, the aadA gene is fused toa chloroplast transit peptide. In particular embodiments, aadA comprisesSEQ ID NO:2.

In certain embodiments, the explant is further defined as having beenmaintained prior to step (b) under conditions wherein the explant doesnot germinate and remains viable and competent for genetictransformation. In some embodiments said conditions comprise dehydratingthe explant or a seed comprising the explant. In certain embodiments,the method is further defined as comprising increasing the moisturecontent of the explant prior to or concurrently with step (b). Inparticular embodiments, said conditions comprise an internal moisturecontent of the explant of from about 3% to about 25%. In more particularembodiments, said conditions comprise an internal moisture content ofthe explant of from about 3% to about 16%. In some embodiments saidconditions comprise maintaining the explant at a temperature of betweenabout −80° C. and about 60° C.

In some embodiments, the method comprises priming the explant prior tostep (b). In particular embodiments, priming the seed comprisescontacting the explant or a seed comprising the explant with an aqueoussolution comprising water, a plant growth regulator, a selection agent,or a cell membrane conditioner.

In certain embodiments comprising the method of producing a transgenicplant comprising: (a) transforming at least a first seed explant with aheterologous nucleic acid sequence comprising a selectable markerconferring tolerance to spectinomycin; and (b) regenerating a transgenicplant from the transformed cells, wherein the explant is contacted,prior to, concurrently with, and/or following step (a) or step (b), withat least a first media comprising spectinomycin to select transformedcells comprising said selectable marker, the method further comprisestransforming at least a first cell of the explant with a heterologousnucleic acid is carried out by bacterially-mediated transformation ormicroprojectile bombardment.

In some embodiments, the explant is further defined as having beenexcised from a seed comprising 3% to 25% internal moisture content, or ahydrated or germinating seed comprising 26% to 80% internal moisturecontent, or comprises a tissue of the group consisting of: meristem,immature embryo, embryo, embryonic axis, cotyledon, hypocotyl,mesocotyl, leaf, primary leaf base, leaf disc, shoot tip, and plumule.In certain embodiments, the explant is further defined as having beenexcised from a germinated or imbibed seed. In other embodiments, theexplant is not contacted with a media comprising spectinomycinsubsequent to step (a). In particular embodiments, the first media is aliquid. In other embodiments, one or more of steps (a)-(b) areautomated.

In another aspect, the invention provides a nucleic acid constructcomprising two sequences conferring resistance to spectinomycin orstreptomycin, wherein the first sequence is operably linked to apromoter active in a plant cell, and the second sequence is operablylinked to a promoter active in a prokaryotic cell. In a particularembodiment, the sequences conferring resistance to spectinomycin orstreptomycin encode a polypeptide comprisingaminoglycoside-3′-adenyltransferase (aadA) activity. In a moreparticular embodiment, at least one of the sequences comprises SEQ IDNO:1 or SEQ ID NO:2.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings are part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to thedrawing in combination with the detailed description of specificembodiments presented herein.

FIG. 1: Magnified details of four soy explants from treatments withdifferent levels of TDZ added to the inoculum/co-cultivation medium.Each explant developed de novo buds/shoots (bottom) and some wereGFP-positive (top). The pictures on the top row were taken in amicroscope with a modified blue light source that detects GFP-expressingtissue by fluorescence. The same images were taken with a standard whitelight source to show all the developed buds/shoots (bottom row).

FIG. 2: Plasmid map of pMON96999.

FIG. 3: Outline of spectinomycin selection protocol “A”. Selection,shoot induction and elongation on liquid or semi-solid medium; rootingdetached shoots on semi-solid medium.

FIG. 4: Outline of spectinomycin selection protocol “B”. Selection,shoot induction, and elongation on liquid or semi-solid medium; rootingdetached shoots in OASIS plugs with liquid medium without selection.

FIG. 5: Outline of spectinomycin selection protocols “C” and “D”(bottom), and comparison with protocol for selection using glyphosate(top). For Protocol “C,” after co-culture the explants are retained inthe original PLANTCONs and 12 ml of liquid selection medium is added.Four days later, they are transferred onto semi-solid selection mediumfor selection and shoot induction. For Protocol “D,” after co-culture,the explants are directly transferred onto the semisolid medium forselection and shoot induction. In both protocols “C” and “D”, theexplants producing green shoots are moved to Oasis® plugs with liquidmedium without selection for shoot elongation and root induction. Forprotocol using selection with glyphosate (top), wherein shoot induction,and shoot elongation is on semi-solid medium with selection, rooting ofdetached shoots is also performed on semi-solid medium with selection.

FIG. 6: Plasmid map of pMON107379 comprising 2 T-DNAs, OriRi, and aadA.

DETAILED DESCRIPTION OF THE INVENTION

The following is a detailed description of the invention provided to aidthose skilled in the art in practicing the present invention. Those ofordinary skill in the art may make modifications and variations in theembodiments described herein without departing from the spirit or scopeof the present invention.

The invention provides methods and compositions for use of spectinomycinas a selective agent for preparing, screening, transforming, andregenerating explants from soybean, corn, cotton, or canola plants,among others, to obtain transformed plant tissues and plants. In someaspects, various portions of the described methods may be automated,high-throughput procedures. An explant such as a mature or immatureembryo is obtained, for instance from a seed, and may be transformed,for instance via a bacterially-mediated or microprojectile bombardmentapproach. In certain embodiments, at the time that a heterologous DNA iscontacting the explant, or subsequently, the explant is contacted by acytokinin selected from the group consisting of thidiazuron, BAP(6-Benzylaminopurine), kinetin, CPPU(N-(2-Chloro-4-pyridyl)-N′-phenylurea), 2iP (6-(y,y-Dimethylallylamino)purine), Zeatin, Zeatin-riboside, Adenine, and TIBA(2,3,5-Triiodobenzoic acid)) or other agent like dikegulac. Tofacilitate the contacting of an explant with the cytokinin, thecytokinin may be added to the bacterial inoculum to be used in thetransformation prior to the contacting of the explant with the inoculum.In certain embodiments, the cytokinin which is employed is BAP at aconcentration of about 0-3 mg/L or about 0.25-3 mg/L, or TDZ (at about0-3 mg/l or about 0.25-3 mg/L). In certain embodiments, the cytokinin orother agents may also be added during seed imbibition to treat theexplants before they are excised.

Use of spectinomycin, with or without cytokinin treatment, atconcentrations of between 15-1500 mg/L is contemplated, for instanceabout 25, 50, 100, 150, 250, 300, 500, 1000, or 1500 mg/L. If a methodfor bacterially-mediated transformation is used, the spectinomycin maybe added to the bacterial inoculum prior to its contacting the explant.Alternatively, if a bacterially-mediated or microprojectile-mediatedtransformation method is used, the spectinomycin may be added prior to,concurrently, or following the step of transforming a soybean, corn,cotton, or canola cell, so as to select for cells transformed with aheterologous nucleic acid. Spectinomycin may also be employed as a“pulse” for a portion of the period of time for a described tissueculture growth step, such as the pre-culture step, co-cultivation step,delay step, or selection step, and optionally at a higher concentrationof about 1000 mg/L.

The transformation frequencies (“TFs”) obtained using the methods andcompositions described herein have not been achievable in the prior art.Thus, an increase in TF of 2-10 or 5-10 fold (and even higher in somecases) over that found, for instance, when using glyphosate or dicambaas the selective agent for transformation of soybean or cotton, has beenachieved. Additionally, the increased transformation efficiency allowsfor development of an efficient 2 T-DNA transformation system usingspectinomycin selection, thus allowing for stacking of transgenic traitsby transformation and crossing of plants already comprising a transgenictrait with a nucleic acid encoding an additional trait of interest, andthen screening for plants also comprising the nucleic acid encoding theadditional trait.

Combined with an increased TF, the methods described also allow for morerapid regeneration of candidate transformed plant tissues, increasedefficiency in identifying and growing transformed shoots and plants, andreduced costs and ergonomic burden, while simplifying and reducing thelabor necessary to produce transformed plants. For instance, afterspectinomycin resistant shoots with green (i.e. spectinomycin resistant)buds or leaves have elongated and are screenable or scoreable as beingspectinomycin resistant, they may be placed in soil or on a soilsubstitute such as on a rooting medium, in the presence or absence ofthe selective agent. Shoots elongating from such an explant areroutinely shown to be transgenic and give rise to R₁ and subsequentprogeny that are transgenic, while the roots developing from suchexplants may be transgenic or non-transgenic. Thus, a plant comprising atransgenic shoot and a partly or fully non-transgenic root system isalso contemplated. A method for regenerating a whole plant fromtransgenic shoots from transformed meristematic tissue while roots arenon-transgenic, by culturing of transformed tissue on a medium lacking aselective agent, is also contemplated. The described methods thus allowfor a significant decrease in the time spent under selective conditionsand in usage of the selective agent, thus reducing potential costs aswell.

In order to provide a clear and consistent understanding of thespecification and the claims, including the scope given to such terms,the following definitions are provided.

“Embryo” is part of a seed, consisting of precursor tissues(meristematic tissues) for the leaves, stem, and root. Once the embryobegins to grow (germinate), it becomes a seedling plant.

“Meristem” or “meristematic tissue” consists of undifferentiated cells,the meristematic cells, which differentiate to produce multiple plantstructures including stem, roots, leaves, germline tissue and seeds. Themeristematic cells are the targets for transformation to obtaintransgenic plants.

“Explant” is a term used to refer to target material for transformation,comprising meristematic tissue. It may refer to plant tissues including,without limitation, one or more embryos, cotyledons, hypocotyls, leafbases, mesocotyls, plumules, protoplasts, and embryonic axes.

“Chimeric plants” are plants that are composed of tissues that are notgenetically identical, i.e., the plants will have only a portion orfraction of their tissues transformed, whereas the remainder of thetissues are not genetically transformed.

“Germline transformation” occurs when the gene of interest istransformed into cells that give rise to pollen or ovule thus intoseeds.

The explants may be transformed by a selected heterologous DNA sequence,and transgenic plants may be regenerated therefrom, without the need forgenerating a callus cell culture from the transformed explant in orderto obtain transgenic progeny plants. The selected heterologous DNAsequence may for instance encode a screenable or selectable marker,and/or comprise a gene of agronomic interest specifying a trait to beexhibited by a soybean, corn, cotton, or canola plant or cell resultingfrom the expression of the heterologous nucleic acid. The trait may beagronomically useful, for instance resulting in enhanced yield,herbicide tolerance, pest or pathogen resistance, or environmentaladaptability, among other phenotypes. The trait may also specifyproduction of a desired end-product.

Such transformation and regeneration methods allow for a fast andefficient high-throughput process for generating transformed plants.Mechanization significantly reduces the estimated man-hours needed toproduce 10,000 explants, for instance in the case of cotton from about40 to only 2.4 hours, significantly saving labor costs. Such a techniqueallows larger numbers of transgenes to be tested and higher qualityevents to be chosen for further analysis, as only a very small number oftransformation events are expected to exhibit the most desiredexpression profiles suitable for commercial development. A mechanizedexcision process also allows better timing and scheduling oftransformation steps, because of increased flexibility in explantdelivery. Use of a mechanized process for explant excision may providesignificant monetary, safety and flexibility benefits. However, explantpreparation may also be performed manually.

Prior to imbibition, germination, and/or explant excision, seeds may besubjected to a sterilization step as well as a culling step, to avoidmicrobial contamination, to remove seeds with a high degree of bacterialor fungal contamination, and also to remove seeds that may for anyreason be unlikely to produce viable explant tissue for use with thepresent invention. Culling may be carried out, for example, based onparameters such as the size, color, or density of the seed or othercharacteristics, including chemical composition characteristics.Examples of culling methods may include the use of an automatic scaleafter size sorting. An optical sorter suitable for this purpose is theSortex 3000 Series Color Sorter (Buhler-Sortex KK, Yokohama, Japan).Other culling techniques may also be employed including culling bymoisture content. After excision, explants may also be subjected to arehydration or pre-culture step prior to being transformed with aheterologous nucleic acid.

In specific embodiments, excision is mechanically performed usingrollers that crush seeds applied to their faces, which can becounter-rotating. The gap between the rollers may be adjusted based onthe size of the applied seeds. Roller material may, for instance, beelastomeric or metallic. In certain embodiments, stainless steel rollershave been found to retain beneficial working qualities even followingrepeated and sustained use. For use with cotton seeds, rollers withsecondary grooves have been found to efficiently grip and crush seedwith minimal damage to the meristematic explant seed fraction. Methodsfor mechanized excision of plant explants are known, for instance seeU.S. Provisional Patent Application Ser. Nos. 60/894,096 and 60/915,066,and U.S. Patent Application Publication No. 2005/0005321, incorporatedby reference herein in their entirety.

In one embodiment, an explant prepared in accordance with the inventionmay be defined as having an internal moisture of about 4-25%, includingabout 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, 22,23, 24, and 25% internal moisture, and specifically including all rangesderivable between any two such values. In particular embodiments, seedsfrom which explants are to be prepared may be harvested at apredetermined internal moisture suitable for isolating transformablematerial therefrom. In certain non-limiting embodiments, seeds fromwhich explants are obtained may be defined as having an internalmoisture of about 3-25%, including about 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 16, 17, 18, 19, 20, 21, 22, 23, 24, and 25% internalmoisture, and specifically including all ranges derivable between anytwo such values, such as, for example, from about 4% to 16%. In certainembodiments, brittleness of seeds may be altered by manipulatingmoisture content, allowing for efficient splitting of seeds andpreparation of explants. For instance, an internal moisture content suchas 3% to 7% may be advantageous. Seeds may be held at such moisturecontents or any other moisture content yielding stable storageconditions (and transformable explants) prior to use. The seeds incertain embodiments may be soybean, corn, cotton, or canola seeds.

Dry explants (explants that have been excised from seed under lowmoisture conditions) or dried wet explants (explants that have beenexcised from seed following hydration/imbibition and are subsequentlydehydrated and stored) of various ages may be used. In one embodiment,explants are relatively “young” in that they have been removed fromseeds for less than a day, for example, from about 1 to 24 hours, suchas about 2, 3, 5, 7, 10, 12, 15, 20, or 23 hours prior to use. In otherembodiments, explants may be stored for longer periods, including days,weeks, months or even years, depending upon storage conditions used tomaintain explant viability. Those of skill in the art in particular willunderstand that storage times may be optimized such that the qualityand/or yield of transformants as well as the efficiency of thetransformation process is maximized. This can be carried out for anyparticular transformation protocol, for example, such asAgrobacterium-mediated transformation, microprojectile bombardmenttransformation, as well as other transformation procedures.

In some embodiments, a dry seed or an explant may be first primed, forexample, by imbibition of a liquid such as water or a sterilizationliquid, redried, and later used for transformation and regeneration. Inother embodiments, the seed or the explant may be primed by raising theinternal seed moisture content to greater than 30%, holding the seed orthe explant at a time point, and then re-initiating imbibition at alater time point. In an alternative embodiment, the seed or the explantmay be primed by raising the internal moisture content to greater than30%, storing the seed or the explant for a predetermined period, dryingthe seed or the explant to the internal moisture content of below 20%,and then re-initiating imbibition.

Regenerable transformable explants may be harvested that contain no,some, or a part of each cotyledon remaining attached to the embryonictissue, for example as much as ¼ of the cotyledon. These explants areconsidered substantially similar, as they may each result in a stabletransformed plant. The explant should however contain at least some ofthe meristematic region of the embryo such that typically the explantcan produce a shoot within 12 weeks of the onset of tissue culturegrowth conditions.

The explant may be recovered from a hydrated seed, from dry storableseed, from a partial rehydration of dried hydrated explant, wherein“hydration” and “rehydration” is defined as a measurable change ininternal seed moisture percentage, or from a seed that is “primed”; thatis, a seed that has initiated germination but has been appropriatelyplaced in stasis pending favorable conditions to complete thegermination process. Those of skill in the art will be able to usevarious hydration methods and optimize length of incubation time priorto transformation. The resulting novel explant is storable and cangerminate and or be transformed when appropriate conditions areprovided. Thus the new dry, storable meristem explant may be referred toas an artificial seed.

Following excision, one of skill in the art may store the explantaccording to the disclosed methods prior to subsequent use. Methods andparameters for drying, storing, and germinating seed are known in theart (e.g. Senaratna et al., 1983; Vertucci and Roos, 1990; Chai et al.,1998). Storage of excised meristems in accordance with the currentinvention may be carried out using modifications of such storageconditions as desired. Any such conditions may be used as desired,including at temperatures, for example, of from about −80° C. to about60° C. Temperatures of about −20° C. to room temperature in particularhave been found to function well, but the invention is in no way limitedto these temperatures.

The data described in the Examples illustrates, for instance, thatstored seed explants comprising meristematic tissue may remain viableand useful for subsequent genetic transformation and regeneration forweeks or months following excision from seeds (e.g. Example 12).Manipulation of excision, sterilization, storage, hydration,redehydration, and transformation parameters allows development ofefficient automated high throughput plant transformation protocols.Rehydration, priming and hydration conditions are also presented. Atypical protocol for machine excision, may involve placing seeds for 15minutes in a bleach solution of 200 ppm active Cl, followed by a 2 hourperiod of no liquid exposure, followed by an overnight hydration ineither bean germination medium (BGM) or a bleach solution of 50 ppmactive Cl.

A number of parameters for obtaining and handling explants may bevaried. In one embodiment, the excision method may be manual; in analternative embodiment excision occurs by an automated process. In otherembodiments sterilization may be performed by contacting a seed orexplant with a liquid sterilizing agent. The addition to a co-culturemedia (like INO) of nystatin (50 ppm) and thiabendazole (10 ppm)dissolved in DMSO (1.0 ml of DMSO per liter of INO) may improve thehealth of explants, likely by controlling yeasts and fungi commonlyfound in and on seeds and can be a useful tool when performing largeand/or automated tissue culture. In an alternative embodiment, a seed oran explant may be contacted with a gaseous sterilizing agent. In analternative embodiment, a seed or an explant may be contacted with anirradiating sterilizing agent such as UV light. In an alternativeembodiment, a seed or an explant may be sterilized by subjecting theseed or the explant to a brief period of high temperatures so as toreduce the vigor of biological contaminants such as adventitiousbacteria and fungi on the surface of the seed or the explant withoutreducing the vigor of the seed or the explant. This can be achieved at atemperature higher than 40° C.; preferably the temperature is between40° C. to 90° C. The temperature can be raised, for instance, by eitherforced heated air or steam. Such temperatures can be provided by dryersproduced by Bry-Air Inc. (Sunbury, Ohio, USA). In still a furtherembodiment, moisture content of the seed at the time of excision may bevaried. In another embodiment, the temperature of the seed at the timeof excision may be varied. In other embodiments, a storage parameterfollowing excision may be varied. For instance, in one embodiment therelative humidity under which explant storage occurs may be varied. Inanother embodiment, the explant storage temperature may be varied. Inyet other embodiments, the length of explant storage time may vary. Inyet other embodiments, the composition of the medium in which theexplant is stored may vary. Further parameters that may be manipulatedinclude hydration and rehydration media compositions, incubationtemperature, length of time, and transformation methods, among others.

Following excision, the invention also provides methods and apparati forscreening to transformable meristematic explant material fromnon-transformable damaged explants, cotyledons, seed coats, and otherdebris. The methods may be performed manually, or may be partially orfully mechanized. In certain embodiments, the screening process issubstantially mechanized. For instance, one or more steps of sieving maybe performed, using sieves of appropriate size based on size of theseeds being crushed and the explants being isolated. Bulk yield ofcrushed seed that has passed through the rollers may be put through aseries of separation sieves, such that unwanted large and small debrisare separated from the desired explant by size exclusion. This may beeffectively accomplished, for instance with cottonseed material, usingU.S. Standard sieves such as: #8 (2.36 mm opening), #10 (2.0 mmopening), #16 (1.18 mm opening), and others as appropriate (e.g.elongated window sieves such as 1/16″×¾″, 1/18″×¾″, 1/19″×½″, or1/20″×½″). Sieves with other opening sizes may be fabricated as neededfor given seed sizes, based on the size of material being applied. Thelength of time for the screening process and the vigor of sieving mayalso be adjusted to enhance the throughput and/or yield of the process.

Other screening methods may also be utilized, such as by measuringdifferential buoyancy in solutions of explant material versus debris. Afraction of material that floats in an aqueous solution has been foundto be enriched for intact transformable explants. A dry-excised explantmay be utilized. Combinations of such screening methods may also beused. The fraction of material with transformable explants may compriseboth meristematic tissues and other tissues, such as portions ofcotyledons. The explant should however contain at least some of themeristematic region such that typically the explant can produce a bud orshoot within 12 weeks of the onset of appropriate growth conditions.

In certain embodiments the excised and screened tissues may betransformed with a heterologous gene of interest. Various methods havebeen developed for transferring genes into plant tissue including highvelocity microprojection, microinjection, electroporation, direct DNAuptake and, bacterially-mediated transformation. Bacteria known tomediate plant cell transformation include a number of species of theRhizobiaceae, including, but not limited to, Agrobacterium sp.,Sinorhizobium sp., Mesorhizobium sp., and Bradyrhizobium sp. (e.g.Broothaerts et al., 2005; U.S. Patent Application Publication2007/0271627). Targets for such transformation have often beenundifferentiated callus tissues, although differentiated tissue also hasbeen used for transient and stable plant transformation, and may be inthis instance. Co-culture and subsequent steps may be performed in darkconditions, or in the light, e.g. lighted Percival incubators, forinstance for 2 to 5 days (e.g. a photoperiod of 16 hours of light/8hours of dark, with light intensity of ≧5 μE, such as about 5-200 μE orother lighting conditions that allow for normal plastid development) ata temperature of approximately 23 to 25° C., and may be performed at upto about 35° C. or 40° C.

In designing a vector for the transformation process, one or moregenetic components are selected that are introduced into the plant cellor tissue. Genetic components can include any nucleic acid that isintroduced into a plant cell or tissue using the method according to theinvention. In one embodiment, the genetic components are incorporatedinto a DNA composition such as a recombinant, double-stranded plasmid orvector molecule comprising at least one or more of following types ofgenetic components: (a) a promoter that functions in plant cells tocause the production of an RNA sequence, (b) a structural DNA sequencethat causes the production of an RNA sequence that encodes a product ofagronomic utility, and (c) a 3′ non-translated DNA sequence thatfunctions in plant cells to cause the addition of polyadenylatednucleotides to the 3′ end of the RNA sequence.

The vector may contain a number of genetic components to facilitatetransformation of the plant cell or tissue and regulate expression ofthe structural nucleic acid sequence. In one preferred embodiment, thegenetic components are oriented so as to express an mRNA, that in anoptional embodiment can be translated into a protein. The expression ofa plant structural coding sequence (a gene, cDNA, synthetic DNA, orother DNA) that exists in double-stranded form involves transcription ofmessenger RNA (mRNA) from one strand of the DNA by RNA polymerase enzymeand subsequent processing of the mRNA primary transcript inside thenucleus. This processing involves a 3′ non-translated region that addspolyadenylated nucleotides to the 3′ ends of the mRNA. Means forpreparing plasmids or vectors containing the desired genetic componentsare well known in the art.

When a DNA construct contains more than one T-DNA, these T-DNAs and thetransgenes contained within may be integrated into the plant genome atseparate loci. This is referred to as “co-transformation” (U.S. Pat. No.5,731,179, WO 00/18939). The process of co-transformation, where twoT-DNAs are at different loci in the plant genome and therefore segregateindependently in the progeny, can be achieved by delivery of the T-DNAswith a mixture of Agrobacteria transformed with plasmids carrying theseparate T-DNA. Co-transformation can also be achieved by transformingone Agrobacterium strain with two binary DNA constructs, each containingone T-DNA (e.g. Daley et al., 1998). Two T-DNAs may also be designed ona single DNA vector, followed by transforming the vector into a plantcell and then identifying the transgenic cells or plants that haveintegrated the T-DNAs at different loci (U.S. Pat. No. 5,731,179, WO00/18939, Komari et al, 1996; U.S. Pat. No. 7,288,694).

A two T-DNA system is a useful method to segregate the marker gene fromthe agronomically important gene of interest (GOI) in a transgenicplant. The marker gene generally has no further utility after it hasbeen used to select or score for the transformed plant cell. A singleDNA vector carrying the two-T-DNAs is one method to construct a twoT-DNA transformation system. However because of the occurrence of bothT-DNAs on a single DNA construct, both may be transferred into the plantgenome at the same locus. This occurs when one of the border DNAmolecule of the first T-DNA is not recognized during the integrationprocess. This reduced efficiency adds to the cost of producing theevents and selecting for the individuals that have T-DNAs integrated atan independent locus. It thus also may be desirable to have DNAconstructs and a method wherein it is possible to chemically selectagainst individuals that have incorporated the two T-DNAs at the samelocus, while screening for the presence/absence and linkage status ofeach of the T-DNAs.

Transcription of DNA into mRNA is regulated by a region of DNA usuallyreferred to as the “promoter”. The promoter region contains a sequenceof bases that signals RNA polymerase to associate with the DNA and toinitiate the transcription into mRNA using one of the DNA strands as atemplate to make a corresponding complementary strand of RNA. A numberof promoters that are active in plant cells have been described in theliterature. Such promoters would include but are not limited to thenopaline synthase (NOS) and octopine synthase (OCS) promoters that arecarried on Ti plasmids of Agrobacterium tumefaciens, the caulimoviruspromoters such as the cauliflower mosaic virus (CaMV) 19S and 35Spromoters and the Figwort mosaic virus (FMV) 35S promoter, and theenhanced CaMV35S promoter (e35S). A variety of other plant genepromoters that are regulated in response to environmental, hormonal,chemical, and/or developmental signals, also can be used for expressionof heterologous genes in plant cells, including, for instance, promotersregulated by (1) heat (Callis et al., 1988, (2) light (e.g., pea RbcS-3Apromoter, Kuhlemeier et al., (1989); maize RbcS promoter, Schaffner etal., (1991); (3) hormones, such as abscisic acid (Marcotte et al., 1989,(4) wounding (e.g., Wuni, Siebertz et al., 1989); or other signals orchemicals. Tissue specific expression is also known. As described below,it is preferred that the particular promoter selected should be capableof causing sufficient expression to result in the production of aneffective amount of the gene product of interest. Examples describingsuch promoters include without limitation U.S. Pat. No. 6,437,217 (maizeRS81 promoter), U.S. Pat. No. 5,641,876 (rice actin promoter), U.S. Pat.No. 6,426,446 (maize RS324 promoter), U.S. Pat. No. 6,429,362 (maizePR-1 promoter), U.S. Pat. No. 6,232,526 (maize A3 promoter), U.S. Pat.No. 6,177,611 (constitutive maize promoters), U.S. Pat. Nos. 5,322,938,5,352,605, 5,359,142 and 5,530,196 (35S promoter), U.S. Pat. No.6,433,252 (maize L3 oleosin promoter), U.S. Pat. No. 6,429,357 (riceactin 2 promoter as well as a rice actin 2 intron), U.S. Pat. No.5,837,848 (root specific promoter), U.S. Pat. No. 6,294,714 (lightinducible promoters), U.S. Pat. No. 6,140,078 (salt induciblepromoters), U.S. Pat. No. 6,252,138 (pathogen inducible promoters), U.S.Pat. No. 6,175,060 (phosphorus deficiency inducible promoters), U.S.Pat. No. 6,635,806 (gamma-coixin promoter), and U.S. patent applicationSer. No. 09/757,089 (maize chloroplast aldolase promoter). Additionalpromoters that may find use are a nopaline synthase (NOS) promoter(Ebert et al., 1987), the octopine synthase (OCS) promoter (which iscarried on tumor-inducing plasmids of Agrobacterium tumefaciens), thecaulimovirus promoters such as the cauliflower mosaic virus (CaMV) 19Spromoter (Lawton et al., 1987), the CaMV 35S promoter (Odell et al.,1985), the figwort mosaic virus 35S-promoter (Walker et al., 1987; U.S.Pat. Nos. 6,051,753; 5,378,619), the sucrose synthase promoter (Yang etal., 1990), the R gene complex promoter (Chandler et al., 1989), and thechlorophyll a/b binding protein gene promoter, PC1SV (U.S. Pat. No.5,850,019), and AGRtu.nos (GenBank Accession V00087; Depicker et al,1982; Bevan et al., 1983) promoters.

Promoter hybrids can also be constructed to enhance transcriptionalactivity (U.S. Pat. No. 5,106,739), or to combine desiredtranscriptional activity, inducibility and tissue specificity ordevelopmental specificity. Promoters that function in plants include butare not limited to promoters that are inducible, viral, synthetic,constitutive as described, and temporally regulated, spatiallyregulated, and spatio-temporally regulated. Other promoters that aretissue-enhanced, tissue-specific, or developmentally regulated are alsoknown in the art and envisioned to have utility in the practice of thisinvention.

The promoters used in the DNA constructs (i.e. chimeric/recombinantplant genes) of the present invention may be modified, if desired, toaffect their control characteristics. Promoters can be derived by meansof ligation with operator regions, random or controlled mutagenesis,etc. Furthermore, the promoters may be altered to contain multiple“enhancer sequences” to assist in elevating gene expression.

The mRNA produced by a DNA construct of the present invention may alsocontain a 5′ non-translated leader sequence. This sequence can bederived from the promoter selected to express the gene and can bespecifically modified so as to increase or decrease translation of themRNA. The 5′ non-translated regions can also be obtained from viralRNAs, from suitable eukaryotic genes, or from a synthetic gene sequence.Such “enhancer” sequences may be desirable to increase or alter thetranslational efficiency of the resultant mRNA. The present invention isnot limited to constructs wherein the non-translated region is derivedfrom both the 5′ non-translated sequence that accompanies the promotersequence. Rather, the non-translated leader sequence can be derived fromunrelated promoters or genes (see, for example U.S. Pat. No. 5,362,865).Examples of non-translation leader sequences include maize and petuniaheat shock protein leaders (U.S. Pat. No. 5,362,865), plant virus coatprotein leaders, plant rubisco leaders, GmHsp (U.S. Pat. No. 5,659,122),PhDnaK (U.S. Pat. No. 5,362,865), AtAnt1, TEV (Carrington and Freed,1990), and AGRtu.nos (GenBank Accession V00087; Bevan et al., 1983).Other genetic components that serve to enhance expression or affecttranscription or translational of a gene are also envisioned as geneticcomponents.

The 3′ non-translated region of the chimeric constructs may contain atranscriptional terminator, or an element having equivalent function,and a polyadenylation signal that functions in plants to cause theaddition of polyadenylated nucleotides to the 3′ end of the RNA. The DNAsequences are referred to herein as transcription-termination regions.The regions are required for efficient polyadenylation of transcribedmessenger RNA (mRNA). RNA polymerase transcribes a coding DNA sequencethrough a site where polyadenylation occurs. Examples of suitable 3′regions are (1) the 3′ transcribed, non-translated regions containingthe polyadenylation signal of Agrobacterium tumor-inducing (Ti) plasmidgenes, such as the nopaline synthase (NOS; Fraley et al., 1983) gene,and (2) plant genes such as the soybean storage protein genes and thesmall subunit of the ribulose-1,5-bisphosphate carboxylase (ssRUBISCO)gene. An example of a preferred 3′ region is that from the ssRUBISCO E9gene from pea (European Patent Application 0385 962).

In one embodiment, the vector contains a selectable, screenable, orscoreable marker gene. These genetic components are also referred toherein as functional genetic components, as they produce a product thatserves a function in the identification of a transformed plant, or aproduct of agronomic utility. The DNA that serves as a selection orscreening device may function in a regenerable plant tissue to produce acompound that would confer upon the plant tissue resistance to anotherwise toxic compound. A number of screenable or selectable markergenes are known in the art and can be used in the present invention.Genes of interest for use as a marker would include but are not limitedto GUS, green fluorescent protein (GFP), luciferase (LUX), among others.In certain embodiments, the vector comprises an aadA gene withassociated regulatory elements encoding resistance to spectinomycin inplant cells. In a particular embodiment, the aadA gene comprises achloroplast transit peptide (CTP) sequence that directs the transport ofthe AadA gene product to the chloroplast of a transformed plant cell. Inother embodiments, the vector comprises a spectinomycin resistance genewith appropriate regulatory elements designed for expression in abacterial cell, such as an Agrobacterium cell, so that the selectionreagent may be added to a co-cultivation medium, and allowing obtentionof transgenic plants for instance without further use of the selectiveagent after the co-culture period.

The present invention can be used with any suitable plant transformationplasmid or vector containing a selectable or screenable marker andassociated regulatory elements as described, along with one or morenucleic acids expressed in a manner sufficient to confer a particulardesirable trait. Examples of suitable structural genes of agronomicinterest envisioned by the present invention would include but are notlimited to genes for disease, insect, or pest tolerance, herbicidetolerance, genes for quality improvements such as yield, nutritionalenhancements, environmental or stress tolerances, or any desirablechanges in plant physiology, growth, development, morphology or plantproduct(s) including starch production (U.S. Pat. Nos. 6,538,181;6,538,179; 6,538,178; 5,750,876; 6,476,295), modified oils production(U.S. Pat. Nos. 6,444,876; 6,426,447; 6,380,462), high oil production(U.S. Pat. Nos. 6,495,739; 5,608,149; 6,483,008; 6,476,295), modifiedfatty acid content (U.S. Pat. Nos. 6,828,475; 6,822,141; 6,770,465;6,706,950; 6,660,849; 6,596,538; 6,589,767; 6,537,750; 6,489,461;6,459,018), high protein production (U.S. Pat. No. 6,380,466), fruitripening (U.S. Pat. No. 5,512,466), enhanced animal and human nutrition(U.S. Pat. Nos. 6,723,837; 6,653,530; 6,5412,59; 5,985,605; 6,171,640),biopolymers (U.S. Pat. Nos. RE37,543; 6,228,623; 5,958,745 and U.S.Patent Publication No. 20030028917). Also environmental stressresistance (U.S. Pat. No. 6,072,103), pharmaceutical peptides andsecretable peptides (U.S. Pat. Nos. 6,812,379; 6,774,283; 6,140,075;6,080,560), improved processing traits (U.S. Pat. No. 6,476,295),improved digestibility (U.S. Pat. No. 6,531,648) low raffinose (U.S.Pat. No. 6,166,292), industrial enzyme production (U.S. Pat. No.5,543,576), improved flavor (U.S. Pat. No. 6,011,199), nitrogen fixation(U.S. Pat. No. 5,229,114), hybrid seed production (U.S. Pat. No.5,689,041), fiber production (U.S. Pat. Nos. 6,576,818; 6,271,443;5,981,834; 5,869,720) and biofuel production (U.S. Pat. No. 5,998,700).Any of these or other genetic elements, methods, and transgenes may beused with the invention as will be appreciated by those of skill in theart in view of the instant disclosure.

Alternatively, the DNA sequences of interest can affect these phenotypesby encoding a an RNA molecule that causes the targeted inhibition ofexpression of an endogenous gene via gene silencing technologies such asantisense-, co-suppression-mediated mechanisms, RNAi technologiesincluding miRNA (e.g., U.S. Patent Application Publication2006/0200878).

Exemplary nucleic acids that may be introduced by the methodsencompassed by the present invention include, for example, DNA sequencesor genes from another species, or even genes or sequences that originatewith or are present in the same species, but are incorporated intorecipient cells by genetic engineering methods rather than classicalreproduction or breeding techniques. However, the term “exogenous” isalso intended to refer to genes that are not normally present in thecell being transformed, or perhaps simply not present in the form,structure, etc., as found in the transforming DNA segment or gene, orgenes that are normally present yet that one desires, e.g., to haveover-expressed. Thus, the term “exogenous” gene or DNA is intended torefer to any gene or DNA segment that is introduced into a recipientcell, regardless of whether a similar gene may already be present insuch a cell. The type of DNA included in the exogenous DNA can includeDNA that is already present in the plant cell, DNA from another plant,DNA from a different organism, or a DNA generated externally, such as aDNA sequence containing an antisense message of a gene, or a DNAsequence encoding a synthetic or modified version of a gene.

In one embodiment, transformation of plant tissue is performed by abacterially-mediated method, such as an Agrobacterium or otherRhizobia-mediated method, and the DNA sequences of interest are presenton one or more T-DNAs (U.S. Pat. Nos. 6,265,638, 5,731,179; U.S. PatentApplication Publications 2005/0183170; 2003110532) or other sequence(e.g., vector backbone) that is transferred into a plant cell. TheT-DNAs may be bound by RB and/or LB sequences or may have no bordersequences. The sequences that may be transferred into a plant cell maybe present on one transformation vector in a bacterial strain beingutilized for transformation. In another embodiment, the sequences may bepresent on separate transformation vectors in the bacterial strain. Inyet another embodiment, the sequences may be found in separate bacterialcells or strains used together for transformation.

The DNA constructs used for transformation in the methods of presentinvention may also contain the plasmid backbone DNA segments thatprovide replication function and antibiotic selection in bacterialcells, for example, an Escherichia coli origin of replication such asori322, a broad host range origin of replication such as oriV or oriRi,and a coding region for a selectable marker such as Spec/Strp thatencodes for aminoglycoside adenyltransferase (aadA) conferringresistance to spectinomycin or streptomycin (e.g. U.S. Pat. No.5,217,902; or Sandvang, 1999). For plant transformation, the hostbacterial strain is often Agrobacterium tumefaciens ABI, C58, LBA4404,EHA101, or EHA105 carrying a plasmid having a transfer function for theexpression unit. Other strains known to those skilled in the art ofplant transformation can function in the present invention.

Bacterially-mediated gene delivery (e.g. Agrobacterium-mediated; U.S.Pat. Nos. 5,563,055; 5,591,616; 5,693,512; 5,824,877; 5,981,840) can bemade into cells in the living meristem of an embryo excised from a seed(e.g. U.S. Pat. No. 6,384,301), and the meristematic region may becultured in the presence of a selection agent such as spectinomycin. Theresult of this step is the termination or at least growth retardation ofmost of the cells into which the foreign genetic construction has notbeen delivered with the simultaneous formation of shoots, which arisefrom a single transformed meristematic cell, or small cluster of cellsincluding transformed meristematic cells. In particular embodiments, themeristem can be cultivated in the presence of spectinomycin,streptomycin or other selective agent, tolerance to which is encoded bythe aadA gene. Examples of various selectable markers and genesproviding resistance against them are disclosed in Miki and McHugh,2004.

In light of this disclosure, numerous other possible regulatoryelements, and other sequences of interest will be apparent to those ofskill in the art. Therefore, the foregoing discussion is intended to beexemplary rather than exhaustive.

Screenable or scorable markers can be employed to identify transgenicsectors/and or plants. Exemplary markers are known and includeβ-glucuronidase (GUS) that encodes an enzyme for various chromogenicsubstrates (Jefferson et al., 1987a; Jefferson et al., 1987b); anR-locus gene, that encodes a product that regulates the production ofanthocyanin pigments (red color) in plant tissues (Dellaporta et al.,1988); a β-lactamase gene (Sutcliffe et al., 1978); a gene that encodesan enzyme for that various chromogenic substrates are known (e.g.,PADAC, a chromogenic cephalosporin); a luciferase gene (Ow et al.,1986); a xy1E gene (Zukowsky et al., 1983) that encodes a catecholdioxygenase that can convert chromogenic catechols; an α-amylase gene(Ikatu et al., 1990); a tyrosinase gene (Katz et al., 1983) that encodesan enzyme capable of oxidizing tyrosine to DOPA and dopaquinone that inturn condenses to melanin; green fluorescence protein (Elliot et al.,1999) and an α-galactosidase. As is well known in the art, other methodsfor plant transformation may be utilized, for instance as described byMiki et al., (1993), including use of microprojectile bombardment (e.g.U.S. Pat. No. 5,914,451; McCabe et al., 1991; U.S. Pat. Nos. 5,015,580;5,550,318; 5,538,880).

A variety of tissue culture media are known that, when supplementedappropriately, support plant tissue growth and development, includingformation of mature plants from excised meristems. These tissue culturemedia can either be purchased as a commercial preparation or customprepared and modified by those of skill in the art. Examples of suchmedia include, but are not limited to those described by Murashige andSkoog, (1962); Chu et al., (1975); Linsmaier and Skoog, (1965); Uchimiyaand Murashige, (1962); Gamborg et al., (1968); Duncan et al., (1985);McCown and Lloyd, (1981); Nitsch and Nitsch (1969); and Schenk andHildebrandt, (1972), or derivations of these media supplementedaccordingly. Those of skill in the art are aware that media and mediasupplements such as nutrients and growth regulators for use intransformation and regeneration are usually optimized for the particulartarget crop or variety of interest. Tissue culture media may besupplemented with carbohydrates such as, but not limited to, glucose,sucrose, maltose, mannose, fructose, lactose, galactose, and/ordextrose, or ratios of carbohydrates. Reagents are commerciallyavailable and can be purchased from a number of suppliers (see, forexample Sigma Chemical Co., St. Louis, Mo.; and PhytoTechnologyLaboratories, Shawnee Mission, Kans.).

Transgenic plants may be regenerated from a transformed plant cell bymethods and compositions disclosed here, such as, but not limited to,spectinomycin Protocols “A” through “D”, as performed on soybean, corn,cotton, or canola explants. A transgenic plant formed usingAgrobacterium transformation methods typically (although not always)contains a single simple recombinant DNA sequence inserted into onechromosome and is referred to as a transgenic event. Such transgenicplants can be referred to as being heterozygous for the insertedexogenous sequence. A transgenic plant homozygous with respect to atransgene can be obtained by sexually mating (selfing) an independentsegregant transgenic plant that contains a single exogenous genesequence to itself, for example an R₀ plant, to produce R1 seed. Onefourth of the R₁ seed produced will be homozygous with respect to thetransgene. Germinating R₁ seed results in plants that can be tested forzygosity, typically using a SNP assay or a thermal amplification assaythat allows for the distinction between heterozygotes and homozygotes(i.e., a zygosity assay).

To confirm the presence of the exogenous DNA or “transgene(s)” in thetransgenic plants a variety of assays may be performed. Such assaysinclude, for example, “molecular biological” assays, such as Southernand northern blotting and PCR™, INVADER assays; “biochemical” assays,such as detecting the presence of a protein product, e.g., byimmunological means (ELISAs and western blots) or by enzymatic function;plant part assays, such as leaf or root assays; and also, by analyzingthe phenotype of the whole regenerated plant.

Once a transgene has been introduced into a plant, that gene can beintroduced into any plant sexually compatible with the first plant bycrossing, without the need for ever directly transforming the secondplant. Therefore, as used herein the term “progeny” denotes theoffspring of any generation of a parent plant prepared in accordancewith the instant invention, wherein the progeny comprises a selected DNAconstruct. A “transgenic plant” may thus be of any generation.“Crossing” a plant to provide a plant line having one or more addedtransgenes or alleles relative to a starting plant line is defined asthe techniques that result in a particular sequence being introducedinto a plant line by crossing a starting line with a donor plant linethat comprises a transgene or allele. To achieve this one could, forexample, perform the following steps: (a) plant seeds of the first(starting line) and second (donor plant line that comprises a desiredtransgene or allele) parent plants; (b) grow the seeds of the first andsecond parent plants into plants that bear flowers; (c) pollinate aflower from the first parent plant with pollen from the second parentplant; and (d) harvest seeds produced on the parent plant bearing thefertilized flower.

The present invention also provides for plant parts or a plant producedby the methods of the present invention. Plant parts, withoutlimitation, include fruit, seed, endosperm, ovule, pollen, leaf, stem,and roots. In a preferred embodiment of the present invention, the plantpart is a seed.

In another aspect, the invention provides an isolated nucleic acidmolecule comprising a sequence that encodes a polypeptide comprising achloroplast transit peptide (CTP)-aadA translational fusion. In certainembodiments, the nucleic acid comprises SEQ ID NO:2.

EXAMPLES

Those of skill in the art will appreciate the many advantages of themethods and compositions provided by the present invention. Thefollowing examples are included to demonstrate the preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples that follow representtechniques discovered by the inventors to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments that are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention. All references cited herein are incorporated herein byreference to the extent that they supplement, explain, provide abackground for, or teach methodology, techniques, or compositionsemployed herein.

Example 1 Preparation of Explant and Inoculation Material

A. Soybean

In order to obtain meristematic explant material, soybean seeds (e.g.cv. A3525; Asgrow Seed Company; were processed to separate the embryo,comprising meristematic tissues, from other tissues including the seedcoat and cotyledon(s). Manual preparation of explants provides tissuewhich is suitable for Agrobacterium-mediated transformation of soybeanmeristems (U.S. Pat. No. 6,384,301), and Particle mediatedtransformation methods (U.S. Pat. No. 5,914,451) are known. Mechanicalmethods of extracting explants have also been described in U.S. PatentApplication Publication 20050005321 and U.S. Patent ApplicationPublication 20060059589. All of these methods result in a meristemexplant that is sufficiently transformable by the described methods.

B. Cotton

Cotton seeds were mechanically processed to excise and isolate theirmeristematic tissues. Alternatively, cotton explants may be prepared byexcision of the embryonic axis from the seed, cotyledons, and hypocotyl(e.g. McCabe and Martinell, 1993). In order to obtain transformablemeristematic explant material, cotton seeds (e.g. from genotypes STN474(Stoneville Pedigreed Seed Co., Stoneville, Miss.), Delta Pearl (Deltaand Pine Land Co., Scott, Miss.), DP5415, DP393, 00S04 (Delta and PineLand Co.), SureGrow501 or SureGrow747 (Sure Grow Cotton Seed Company,Maricopa, Ariz.) were processed as follows to separate the embryo,comprising meristematic tissues, from the seed coat and cotyledon(s).Cotton seeds were removed from storage at 4° C. or −20° C. and broughtto room temperature. Seeds were weighed out, placed into a sterilegerminator unit, and surface-sterilized in 50% Clorox (sodiumhypochlorite) for 5 min. Seeds were then rinsed 3 times with steriledistilled water and were hydrated in a liquid hydration medium (CSM) at28° C. in the dark for about 18 hrs (range of 14 to 42 hours).Alternatively, the germination temperature may be lower, for instanceabout 23° C. The CSM medium contained 200 mg/L carbenicillin(PhyoTechnology Laboratories, Shawnee Mission, Kans.), 125 mg/Lcefotaxime (Midwest Scientific, St. Louis, Mo.), 30 mg/L BRAVO 75(Carlin, Milwaukee, Wis.) and 30 mg/L Captan 50 (Carlin). Othersolutions have also successfully been used to hydrate the cotton seeds,including sterile deionized water or water containing a weakconcentration of bleach typically 50 to 1000 ppm sodium hypochlorite.Following hydration, seeds may be used immediately, or stored atrefrigeration temperatures for up to a week prior to further processing.Mechanical excision of cotton explants may also be utilized (WO92/15675;Keller et al., 1997; McCabe & Martinell, 1993; U.S. Patent Publication2005/0005321).

C. Preparation of Agrobacterium for Inoculation and Co-Cultivation

Agrobacterium strain C58 containing a binary vector with one or twoplant expression cassettes as described above was inoculated, from aglycerol stock, into a liquid LB medium (10 g/L sodium chloride, 5 g/Lyeast extract, 10 g/L bacto-tryptone) containing 75 mg/mL spectinomycinand 50 mg/mL kanamycin. The liquid culture was allowed to grow at 28° C.at 200 rpm on a rotary shaker overnight. After the optical density(OD₆₆₀) of the overnight culture reached the target range of 0.4-1.2,the bacterial culture was centrifuged at 3500 rpm for approximately20-25 min to pellet the cells.

Following removal of the supernatant, the pellet was re-suspended in 10mL of an inoculation medium (INO, Table 1). The OD₆₆₀ (an indirectmeasurement of bacterial concentration) was measured and diluted andadjusted to OD₆₆₀ about 0.28-0.32. Once prepared Agrobacterium culturesare prepared, plant explants are exposed to the inoculum, brieflyexposed to sonication energy from a standard laboratory water bathcleaning sonicator such as L&R Ultrasonics QS140 (L&R Manufacturing Co.,Kearny, N.J.); or a Honda W113 sonicator (Honda, Denshi Japan) for 20seconds to 2 minutes, depending on explant type. After the briefsonication step, explants are drained of originating inoculum andtransferred to fresh PLANTCONs each containing 5 ml of INO media and onepiece of filter paper, usually within several hours after commencementof transfection. Explants are then incubated in a lighted chamber(generally 16 hours of light at ≧5 uE) at approximately 23 to 28 C for 1to 5 days. A series of transient GUS expression studies showed that aninoculum OD₆₆₀ of 0.3-0.8 yielded a comparatively higher proportion ofmeristematic transformation and transgene expression. Although lower andhigher OD₆₆₀ measurements also result in successful experimentaloutcomes.

TABLE 1 Composition of inoculation medium. Ingredient Amount/L Magnesiumsulfate (Fisher M63) 0.1 g Ammonium sulfate (Fisher A702) 53.6 mg Sodiumphosphate monohydrate (Fisher S369-500) 60 mg Calcium chloride (SigmaC-3881) 60 mg Boric acid (Fisher A73-3) 0.3 mg Manganese sulfate (SigmaI-2550) 1 mg Zinc sulfate heptahydrate (Sigma Z-1001) 0.2 mg Potassiumiodide (Sigma P-8166) 0.075 mg Sodium Molybdate dihydrate (Sigma S-6646)0.025 mg Cupric sulfate (Fisher C493-500) 2.5 μg Cobalt chloridehexahydrate (Sigma C-2911) 2.5 μg Sequestrene (Ciba 964603) 2.8 mgPotassium nitrate (Sigma P-8291) 1 g Glucose (Phytotech G386) 30 g MES(Sigma M8250) 3.9 g Bring volume to 1 L with de-ionized distilled waterpH with KOH to 5.4 Autoclave Add sterile vitamin stock containing thefollowing Myo-inositol (Sigma I-3011) 10 mg Nicotinic acid (SigmaN-0765) 0.1 mg Pyridoxine HCl (Sigma P-8666) 0.1 mg Thiamine HCl (SigmaT-3902) 1 mg

Example 2 Transformation of Soybean Explants—Treatment with Cytokinin

For Agrobacterium-mediated transformation of soybean, an inoculum wasprepared of strain ABI (C58) harboring a binary vector, such aspMON96999 containing a gus marker gene and an aadA gene conferringresistance to spectinomycin, pMON101343 containing CP4 EPSPS and GUSgenes, or pMON77404 containing a gfp marker gene and a gene encoding CP4EPSPS conferring tolerance to glyphosate, or pMON73737 containing a gfpmarker gene and a DMO gene conferring tolerance to dicamba.

pMON96999 (FIG. 2) contains the uidA gene under the control of anenhanced CaMV35S promoter (U.S. Pat. Nos. 5,322,938; 5,352,605;5,359,142; and 5,530,1960, a 35S leader sequence, and a 3′non-translated region of the nopaline synthase gene from Agrobacteriumtumefaciens (Genbank Accession E01312), and a nuclear-targeted aadA genefor conferring resistance to spectinomycin (U.S. Pat. No. 5,217,902;(SEQ ID NO:1). The aadA adenylyltransferase gene product was targeted tothe chloroplast by a chloroplast transit peptide of Arabidopsis EPSPS(ShkG-CTP2 Klee et al., 1987.), and was under the control of thepromoter for Arabidopsis elongation factor EF-1alpha (Tsf1; US PatentApplication 20050022261) with an FMV-35S enhancer, a Tsf1 leader (exon1), a Tsf1 intron, and a 3′ non-translated region of the pea rbcS2.

Addition of a cytokinin, thidiazuron (TDZ) or BAP at severalconcentrations, was tested during inoculation/co-cultivation, as well asafter. After inoculation, co-cultivation was carried out for about 2-5days (e.g. 3 days) in a Percival incubator at about 23° C. with a 16hour light/8 hour dark photoperiod (light intensity≧5 μE). Thus theresponse of explants to the cytokinin was tested, and the effects of thedifferent treatments on multiple shoot induction and transgenic eventproduction were evaluated.

A. Effects of Cytokinin Treatment with Use of Glyphosate or Dicamba asSelective Agent.

For TDZ treatment during inoculation and co-cultivation, the soybeanexplants were inoculated with Agrobacterium strain ABI harboringpMON77404 (containing genes encoding CP4 EPSPS and GFP), or pMON101343(containing CP4 EPSPS and GUS), and co-cultivated with the inoculumsupplemented with different levels of the cytokinin for 3 days. Afterco-cultivation, the explants were transferred to the selection medium(WPM; Table 2) containing 200 mg/L each of carbenicillin and cefotaxime,100 mg/L Timentin to inhibit growth of Agrobacterium and othercontaminants, and 75 μM glyphosate or 0.01 mg/L dicamba for selection.About twenty-three to thirty days later, the explants were examinedunder a microscope equipped with a filter set for detectingGFP-expressing tissue, or examined for GUS expression, as appropriate.As shown in Table 3, more explants treated with TDZ had developedGFP-expressing buds or young shoots compared with untreated explants.The effect is concentration-related. In this instance, the selectablemarker that was utilized conferred tolerance to glyphosate.

TABLE 2 Composition of WPM used for soybean transformation withglyphosate selection; for dicamba selection, glyphosate was replacedwith 0.01 mg/L dicamba. Ingredient Amount/L LM WPM with vitamins(Phytotech L449) 2.41 g Sucrose (Phytotech S391) 20 g Calcium gluconate(Sigma G-4625) 1.29 g With or without Clearys 3336 WP (Carlin 10-032)0.03 g AGARGEL (Sigma A-3301) 4 g Fill water to 1 L pH 5.6 AutoclaveCarbenicillin (Phytotech C346) (40 mg/mL) 5 mL Cefotaxime (MidwestNDC0039-0019-10) (50 mg/mL) 4 mL Timentin (100 mg/ml) (Duchefa T0190) 1ml Glyphosate (0.5M) 3 mL

TABLE 3 Effect of TDZ treatments during inoculation and co-cultivationon development of transgenic (GFP-positive) bud/shoot development insoybean transformation using glyphosate selection. TDZ level ininoculation and co- # Explants cultivation medium # Explants with GFP+Exp-Trt (mg/L) examined buds/shoots¹ Frequency 1033-1 0 200 27 13.5%  1033-2 0.5 200 36 18% 1033-3 1.0 200 48 24% 1033-4 1.5 200 50 25% 1033-52.0 200 60 30% 1033-6 3.0 200 64 32%Similar results were observed when soybean explants were treated withTDZ after inoculation and co-cultivation, e.g. during the “delay” orselection phase of tissue culture.

However, additional experiments demonstrated that use of TDZ (e.g.during inoculation/co-culture) with glyphosate as the selective agentresulted in a decrease in the number of transformed rooted soybeanshoots, relative to the number obtained in the absence of TDZ, as shownin Table 4.

TABLE 4 Transformation results from glyphosate selection experimentscomparing different TDZ levels in inoculation/co-culture medium. ShootsRooted shoots TDZ for harvested % inoculation/ # Explants % TransformedExpt. co-culture left for Total Shooting Total rooted # (mg/L) harvest #Frequency # shoots 1041 0 435 57 13.1 9 2.1 0.5 456 21 4.6 5 1.1 1 45022 4.9 6 1.3 1092 0 1125 189 16.8 40 3.6 0.5 1200 127 10.6 17 1.4 1 135795 7 22 1.6 1093 0 1000 101 10.1 22 2.2 1 1100 39 3.5 6 0.5 2 1244 181.4 1 0.1 1103 0 900 171 19 53 5.9 2 750 37 4.9 10 1.3 3 850 40 4.7 50.6 1104 0 1050 138 13.1 48 4.6 2 1125 57 5.1 10 0.9 3 1074 20 1.9 8 0.71111 0.5 600 112 18.7 26 4.3 1 600 78 13 19 3.2 2 600 40 6.7 3 0.5 11130.5 1000 55 5.5 14 1.4 1 1000 68 6.8 10 1 2 950 18 1.9 5 0.5

The effect of cytokinin treatment on transformation frequency was alsoassessed using DNA constructs encoding tolerance to another selectiveagent, dicamba. pMON73737, encoding GFP and DMO genes, was utilized.After co-cultivation, the explants were cultured for 4 days on a mediumcontaining 0.01 mg/L dicamba, with or without BAP or TDZ as shown inTable 5. Treatment with either BAP or TDZ resulted in an increase in thenumber of explants displaying GFP positive buds at an early stage suchas 24 days after inoculation (“DAI”, Table 5).

TABLE 5 Effect of BAP and TDZ treatments for 4 days after co-cultivationon development of transgenic (GFP-positive) bud development in soybeantransformation using dicamba selection. Pre-treatment Experiment- w/ BAPor # Explants # Explants w/ Treatment TDZ (4 Days) examined (24 DAI)GFP+ buds (%) 906-1 No pre-treatment 284 6 (2.1%) 906-2 1 mg/L BAP 32512 (3.7%)  906-3 2 mg/L BAP 336 11 (3.3%)  906-4 1 mg/L TDZ 387 71(18.3%) 906-5 2 mg/L TDZ 383 53 (13.8%)

The explants treated with TDZ did not display strong apical dominanceand produced more shoots (de novo multiple shoots) as observed invarious experiments. In contrast, untreated explants showed more growthof the primary shoots (the result of apical dominance) and producedfewer shoots. Those shoots also likely developed from axillary buds.Therefore, the resulting transformation frequency was lower comparedwith the TDZ-treated explants. However, the growth (e.g. elongation) ofthe shoots resulting from transformation of soybean explants undergoingselection on glyphosate or dicamba-containing media was retarded. Usingglyphosate selection, the time from inoculation to subsequenttransformed R1 seed harvest was about 7 months, and the time fordevelopment of transformed rooted shoots was about 10-12 weeks. Additionof a cytokinin (BAP or TDZ) with dicamba selection had no visible effecton final transformation frequencies (TFs) obtained, although it resultedin production of more GFP positive buds at an early stage as shown inTable 5.

B. Effects of Cytokinin Treatment with Use of Spectinomycin as SelectiveAgent

Explants were inoculated with Agrobacterium in inoculation media(Table 1) by sonication for 20 sec, and then cultured on co-culturemedium, which is the same as inoculation medium, as described in Example1, The medium, for both inoculation and co-culture was supplemented with2 mg/L TDZ. Explants were co-cultured for 4 days at about 23° C., with a16 h light/8 h dark photoperiod. After co-culture, 12 ml liquid delaymedium, which is the same as the WPM medium shown in Table 2 except notsolidified with Agargel and lacking a selective agent (glyphosate,dicamba or spectinomycin), was added to each PLANTCON™ (MP Biomedicals,Solon, Ohio) containing the explants. The explants were cultured in thedelay medium for 4 days (28° C., with a 16 h light/8 hr. darkphotoperiod). For selection and shoot induction, the explants weretransferred to the same liquid medium but with addition of differentlevels of spectinomycin (25-250 mg/L spectinomycin). The explants wereindividually implanted into the slits of the foam sponge in the PLANTCONvessel. Each vessel contained 60 ml of medium and one piece of foamsponge holding about 25 explants.

In all treatments, the explants developed multiple buds and shoots.Samples of explants and resulting tissues were collected and assayed forGUS activity at different stages. When explant tissue transformed withan aadA gene and GUS (on pMON96999; FIG. 2) was selected in the presenceof spectinomycin, distinct green (spectinomycin resistant) and bleached(spectinomycin susceptible) buds and shoots were observed, as well asGUS-positive tissues, on a large number of TDZ-treated explantsapproximately 3 weeks after inoculation (Table 8). Up to 80% of theexplants developed GUS-positive buds and shoots within two weeks onselective medium (about 3 weeks after inoculation, i.e. about 22 DAI;Tables 8-9).

TABLE 6 Composition of Agrobacterium co-culture medium 1595, per L.Ingredient Amount TC Water 750 ml B5 stock 1 (see below) 1 ml B5 stock 2(see below) 1 ml B5 stock 3 (see below) 1 ml B5 stock 5 (see below) 1 mlPotassium nitrate (Sigma P-8291) 1 g Glucose (Phytotech G386) 30 g MES(Sigma M-8250) 3.9 g Add TC water to 1 L TC H₂0 to 1000 ml pH with KOHto 5.4 Autoclave Add B5 stock 4 (see below) 1 ml B5 Stock #1 AmmoniumSulfate 53.6 g Magnesium Sulfate 100 g Sodium phosphate Monobasic 60 gB5 Stock #2 Calcium Chloride 60 g B5 Stock #3 Boric Acid 0.30 gManganese Sulfate 1.0 g Zinc Sulfate 0.20 g Potassium Iodide 0.75 gSodium Molybdate 0.025 Copper Sulfate (1 mg/ml stock) 2.5 ml CobaltChloride (1 mg/ml stock) 2.5 ml B5 Stock #4 Thiamine HCl 1.0 g NicotinicAcid 0.1 g Pyridoxine HCl 0.1 g Inositol 10 g B5 Stock #5 Sequestrene2.8 g

A study was also conducted to evaluate if treatment with cytokinin BAPalso enhanced transformation frequency for soybean transformation usingspectinomycin selection by promoting multiple shoots development.Soybean explants were inoculated and co-cultured with Agrobacteriumharboring pMON96999 (FIG. 2). The inoculation and co-culture medium weresupplemented with 0, 1, 2, 3, 4 or 5 mg/L BAP. After co-culture for 3days, the explants were cultured in a delay medium (inoculation mediumlacking selection) for 4 days. The delay medium was also supplementedwith the same level of BAP for each treatment as shown in Table 7. Theexplants were then transferred onto selection medium (the same as thedelay medium but containing 150 mg/L spectinomycin) for shoot inductionand selection. Explants in the treatment without BAP showed more apicaldominance with more elongated primary shoots.

To determine how many explants could develop transformed shoots withoutBAP treatment, explant tissues were assayed for GUS activity at 42 daysafter inoculation. Approximately 18% of the explants had GUS+ buds orsmall shoots (Table 7). Most of them were axillary, and some of themwere apparently chimeric. The explants treated with BAP had much less ornon-elongated primary shoots, and more de novo shoots. Many of theshoots elongated on the selection medium and were harvested to induceroots on the root induction medium also containing 150 mg/Lspectinomycin as in protocol “A”. Transformation frequency (TF) weredetermined based on number of rooted shoots and were shown in Table 7.Since there was only 18% of the explants among the explants not treatedwith BAP that showed GUS+ buds or shoots, a TF much lower than 18% wouldbe expected if the explants were not sacrificed for the GUS assay, sincenot all those GUS+ buds/shoots would continuously develop to eventuallybecome plants. Therefore, the data strongly suggested that BAP treatmentenhanced TF by inhibiting apical dominance and promoting multiple shootdevelopment.

TABLE 7 Effect of BAP treatment during co-culture and post co-culturedelay stage. BAP in co- culture & 4- # Explants d post co- producingculture delay # GUS+ axillary # Exp- medium Explants buds/shoots RootedTrt# (mg/L) left (42DAI) plants² % TF 1118-1¹ 0 352 65 (18.5%) n/a n/a1118-2 1 395 n/a 103 26.1 1118-3 2 347 n/a 94 27.1 1118-4 3 350 n/a 8724.9 1118-5 4 435 n/a 132 30.3 1118-6 5 436 n/a 116 26.6 ¹All theexplants in this treatment were assayed for GUS activity 42 days afterinoculation. ²Rooted in medium containing 150 mg/L spectinomycin.

TABLE 8 Effect of treatments with different levels of TDZ duringinoculation and co-culture on development of GUS-positive buds. TDZlevel for # Explants % Explants with inoculation & assayed for #Explants w/ GUS+ GUS+ buds (13 co-culture GUS buds (13 DAI) DAI) 0.5mg/L 96 18 18.8 1.0 mg/L 96 34 35.4 2.0 mg/L 96 48 50.0

TABLE 9 Percent of explants cultured on medium containing differentlevels of spectinomycin that developed GUS-expressing buds and shoots. #Explants with Experiment- Spectinomycin # Explants GUS+ treatment levelused assayed shoots/buds % 1102-1 25 25 13 52 1102-2 50 25 18 72 1102-3100 25 19 76 1102-4 150 25 18 72 1102-5 200 25 20 80 1102-6 250 25 20 80

Various levels of TDZ were also found to be effective in promotingdevelopment of GUS positive buds. In contrast to studies performed usingglyphosate or dicamba as a selective agent, greenspectinomycin-resistant shoots elongated well, and shoot harvest couldbe performed by six weeks after inoculation. Most cultured explantmaterial produced one elongated shoot at a time, although some producedmore than one shoot. One elongated shoot was harvested from eachtransformed explant. Shoot harvest stopped approximately 9 weeks afterinoculation, although even more elongating shoots were being producedfrom additional explants. Shoots were rooted in a root induction medium(BRM). This medium contained ½ strength of MS salts, MS vitamins, 100mg/l inositol, 100 mg/l cysteine, 30 mg/l sucrose and 100 mg/lticarcillin and was solidified with 8 g/l washed agar and alsosupplemented with 150 mg/L spectinomycin and 0.1 mg/L IAA or 0.25 mg/LIBA as rooting hormone. Spectinomycin was employed at 0-250 ppm, and upto 1000 ppm in some studies. As shown in Table 10, in the first studythe average transformation frequency was 18.6%, ranging from 12.6-26.1%,a significant increase from the approximate 2% transformation frequencyseen in comparable experiments utilizing glyphosate as selective agent.A later study confirmed the result (Table 10). Such high transformationfrequency was found with a wide range of spectinomycin concentrations(25-250 mg/L and up to 1000 mg/L), and 150 mg/L of spectinomycin wastypically used in later studies. Additionally, of the first 32 plantstested to confirm transformation, 31 plants were later shown to betransformed, with only one “escape.”

TABLE 10 Transformation frequency using spectinomycin selection. #Explants Plants left Shoots tested for Experiment- Spec level for shootharvested¹ transformation Treatment (mg/L) harvest Total % SF Total % TF1102-1 25 100 46 46 19 19 1102-2 50 216 101 46.8 48 22.2 1102-3 100 17536 20.6 22 12.6 1102-4 150 111 39 35.1 29 26.1 1102-5 200 188 53 28.2 2714.4 1102-6 250 200 55 27.5 39 19.5 Total 990 330 33.3 184 18.6 1119-150 600 149 24.8 88 14.7 1119-2 100 600 123 20.5 94 15.7 1119-3 150 600125 20.8 110 18.3 1119-4 200 600 91 15.2 75 12.5 1119-5 250 600 108 18.098 16.3 Total 3000 596 19.9 465 15.5 ¹Only one shoot was harvested fromeach explant, although some explants produced multiple shoots. Sinceshoot elongation was not uniform, shoot harvest was stopped 9 weeksafter inoculation although more shoots could be harvested later. “SF” =shoot frequency; “TF” = transformation frequency

Using spectinomycin selection, the time from inoculation to the time fordevelopment of transformed rooted shoots was about 8 weeks, andsubsequent transformed R₁ seed harvest was typically <6 months.

Example 3 Development and Comparison of Rapid Efficient SoybeanTransformation and Culture Protocols Using Spectinomycin Selection

In order to improve the speed and efficiency of soybean transformationusing spectinomycin selection, including with cytokinin treatment,several protocols utilizing spectinomycin selection were compared amongeach other and with a previously employed method that used glyphosate asthe selective agent. Table 11 and FIGS. 3-5 outline protocols used andresults obtained. As noted, the spectinomycin selection protocolsdemonstrated a high frequency of transformation, and a shorter period oftime needed to complete each protocol (inoculation to next generationseed), as compared with the glyphosate selective protocol. Additionalbenefits include simplicity, reduced ergonomic impact, and streamlinedplant handling, leading to lower costs.

The increased frequency in obtaining transgenic plants (˜>10× moreefficient compared to glyphosate or dicamba selection) enables anefficient 2 T-DNA transformation system and method for stacking traitsby transformation into, for example, a ROUNDUP READY® germplasm followedby selection of marker-free segregants of the second gene of interest.Because many soybean breeding lines are themselves glyphosate tolerant,delivering an additional trait into such a genetic background provides asignificant advantage in trait integration.

TABLE 11 Comparison of glyphosate selection protocol with exemplaryspectinomycin selection protocols. Spectinomycin Spectinomycin CP4Selection selection- Spectinomycin Spectinomycin selection- Stepprotocol Protocol A selection-Protocol B selection-Protocol C Protocol DExplant preparation Seed imbibition, explant excision as noted above andin U.S. Patent Publication 20050005321 Inoculation/co- Bulk sonicationBulk sonication or Bulk sonication or Bulk sonication or Bulk sonicationculture sonicated in individual sonicated in individual sonicated inindividual or sonicated in PLANTCON with PLANTCON with TDZ PLANTCON withTDZ individual TDZ or BAP for or BAP for multiple or BAP for multipleshoot PLANTCON multiple shoot shoot induction induction with TDZ orinduction BAP for multiple shoot induction Post co-culture stageSurface-plate explants Add 12 ml liquid Add 12 ml liquid Add 12 mlliquid medium Surface-plate I (shoot on semi-solid selection mediumWPM + CCT medium WPM + CCT WPM + CCT with or explants oninduction/selection medium (WPM + CCT with or without with or withoutwithout spectinomycin semi-solid (carbenicillin, spectinomycin (150mg/L) spectinomycin (150 mg/L) (150 mg/L) into the co- selectioncefotaxime, and into the co- into the co-culture culture PLANTCON tomedium ticarcillin) + 75 μM culture PLANTCON PLANTCON to inhibit inhibitAgrobacterium and (WPM + glyphosate) to inhibit Agrobacterium and startstart selection process if CCT + ~2 weeks Agrobacterium and selectionprocess if spectinomycin is included spectinomycin) start selectionprocess spectinomycin is ~4 days ~4 weeks if spectinomycin is includedincluded ~4 days ~4 days Post co-culture Transfer explants toSurface-plating or Surface-plating or Surface-plating or n/a Stage II(shoot fresh semi-solid implanting explants on implanting explants onimplanting explants on induction and selection medium semi-solidselection semi-solid selection semi-solid selectionelongation/selection) (WPM + CCT + 75 μM medium; or implanting medium;or implanting medium; or implanting glyphosate); implanting explantsinto foam explants into foam explants into foam sponge ~5-6 weeks spongewith slits or in sponge with slits or in with slits or in float on floaton liquid float on liquid selection liquid selection medium selectionmedium medium (WPM + CCT + spec); (WPM + CCT + spec); (WPM + CCT +spec); 4 weeks or longer ~6-7 weeks. ~6-7 weeks. Post co-culture Detachand culture Detach and culture Detach and culture Grow explants w/ greenGrow explants Stage III (rooting, or elongated shoots in elongatedshoots in elongated shoots in shoots in Oasis plugs for w/ green shootsshoot elongation & semi-solid root semi-solid root Oasis plugs w/ simpleshoot elongation and root in Oasis plugs rooting) induction medium w/induction medium w/ liquid medium w/o induction from original for shootglyphosate for root spec for root induction selection.; radicals insimple liquid elongation and induction and and selection. 2-3 weeks ingreenhouse medium w/o selection. root induction selection. 2-3 weeks inlight 2-3 weeks in greenhouse from original ~2-3 weeks in light cultureroom radicals in culture room simple liquid medium w/o selection. 2-3weeks in greenhouse Total duration from 10-12 weeks 9-11 weeks 9-11weeks ~8 weeks ~8 weeks inoculation to obtaining plant Comparison withGlyphosate inhibits Cytokinin (TDZ or BAP) is used duringinoculation/co-culture previous glyphosate apical dominance and toinduce de novo multiple shoots. Visual selection: green resistantselective protocol promotes axillary bud/ putatively transformedbuds/shoots vs. white susceptible shoots; shoot development; whitebuds/shoots stop growth at early stage. Non-visual marker. Putativetransgenic shoots elongate Comparison with n/a Higher transformationHigher TF (~10X); Higher TF (>10X); Higher TF previous glyphosatefrequency (~10X) simplify plant handling shorter cycle; simplify (>10X);shorter selective protocol- system and reduce labor transformation andthe cycle; more benefit and material cost in plant handling system andsimplified greenhouse. reduce labor and material transformation cost ina transformation and plant laboratory and handling greenhouse; reducesystem and ergonomic stress in reduce labor transformation laboratoryand material and greenhouse. cost in transformation laboratory andgreenhouse; reduce ergonomic stress in transformation laboratory andgreenhouse.

Tables 12-13 demonstrate transformation frequencies obtained usingProtocol C or D.

TABLE 12 Soybean transformation frequency using spectinomycin selection,Protocol “C”. Construct # Explant % Explants to # Plant Exp-Trt (pMON) #Explants to the plug the plug handed off % TF 1207-2 96999 (1T) 227 14463.4 70 30.8 1207-4 96999 (1T) 129 89 69.0 47 36.4 1208-3 96999 (1T) 12783 65.4 30 23.6 1208-6 96999 (1T) 192 137 71.4 46 24.0 1216-1 96999 (1T)273 166 60.8 83 30.4 1216-2 96999 (1T) 97 70 72.2 54 55.7 Total 1045 68965.9 330 31.6 1223-1 107379 (2T; OriRi) 293 188 64.2 82 28.0 1223-2107379 (2T; OriRi) 292 167 57.2 65 22.3 1223-3 107379 (2T; OriRi) 254165 65.0 71 28.0 1223-4 107379 (2T; OriRi) 275 191 69.5 72 26.2 Total1114 711 63.8 290 26.0

TABLE 13 Soybean transformation frequency using spectinomycin selection,Protocol “D”. # Explant % Explants to # Plant Exp-Trt Construct (pMON) #Explants to the plug the plug handed off % TF 1224-1 107379 (2T; OriRi)338 208 61.5 74 21.9 1224-2 107379 (2T; OriRi) 234 140 59.8 50 21.41225-1 107379 (2T; OriRi) 235 173 73.6 60 25.5 1225-2 107379 (2T; OriRi)253 178 70.4 65 25.7 1226-1 107379 (2T; OriRi) 205 139 67.8 49 23.91226-2 107379 (2T; OriRi) 201 131 65.2 45 22.4 Total 1466 969 66.1 34323.4 1244-1 107380 (2T; OriV) 264 154 58.3 61 23.1 1244-2 107380 (2T;OriV) 205 124 60.5 50 24.4 1254-1 107380 (2T; OriV) 283 232 82.0 11139.2 1254-2 107380 (2T; OriV) 267 213 79.8 88 33.0 1255-1 107380 (2T;OriV) 202 136 67.3 47 23.3 1255-2 107380 (2T; OriV) 295 198 67.1 94 31.9Total 1516 1057 69.7 451 29.7

Example 4 Comparison of Transformation Frequencies and Event Quality

Several of the above-described spectinomycin transformation protocolswere compared among each other and with the glyphosate selectionprotocol for soybean transformation quality, as shown in Table 14. Inthe table below, “TF” refers to the number of events produced per thenumber of explants; “qTF” refers to the number of quality events pernumber of explants, wherein a quality event is defined as an eventcomprising one copy of the gene-of-interest and lacking backbone (vectorsequence); and “MF TF” refers to the number of events with one copy ofthe gene-of-interest, not linked to marker, and lacking vector backbonesequence, per number of explants subjected to transformation.

TABLE 14 Estimates of transformation quality. # TF % Protocol andExplants # Events (%) +/− # Events quality Estimated vector type testedproduced s.e assayed events qTF % MF TF % Spec Protocol A- 41786 815419.5- 828 19.1 3.7 n/a 1T; OriV (0.89) (0.67) Spec Protocol C- 1045 33031.6  393* 20.9 6.6 n/a 1T; OriV (4.85) (1.09) Spec Protocol C- 1114 29026 286 23.8 6.2 1.8 2T; OriRi (1.34) (0.37) Spec Protocol D- 1516 45129.7 433 20.3 6.0 1.4 2T; OriV (2.69) (0.96) Spec Protocol D- 1466 34323.4 326 24.2 5.7 1.7 2T; OriRi (0.76) (0.29) CP4 Protocol-2T; 21351 5892.76 529 29.9 0.74 0.18 OriV (0.23) (0.08) CP4 Protocol-2T; 21651 3601.66 299 38.8 0.54 0.24 OriRi (0.19) (0.08) *not all events included incalculating TF

The non-linkage rate for estimating MF TF values in Table 14 was basedon the rate estimated by the data in Table 15. As can be seen, certainspectinomycin transformation protocols (“C”, “D”) yielded up to a10-fold increase in the number of “quality events” obtained, as comparedwith the glyphosate selection protocol, while Protocol “A” showed asignificant increase in TF and qTF as well. More than 92% of the eventswere confirmed as germline-transformed, based on GUS expression in R₁immature soybean embryos when using any of Protocols “A”, “C”, or “D”.This “escape” rate is comparable to that found observed from glyphosateselection protocols. The R₀ plants were also found to grow normally andset seeds (R₁ generation) well, averaging almost 200 seeds per plantfrom soybean plants transformed using any of protocols A-D.

TABLE 15 Estimate of non-linkage rate. Selection and # PlantsNon-linkage backbone type assayed rate obtained Spec OriV 122 ~23% SpecOriRi 157 ~29% CP4 OriV 55 ~24% CP4 OriRi 62 ~44%

Example 5 Development of Spectinomycin Transformation System forDry-Excised Soybean Explants

Methods of spectinomycin selection were utilized to transformdry-excised soybean explants, prepared as follows:

1) Dry, viable, seeds (properly stored quality soybean seed isapproximately 10 to 12% internal moisture content) were rinsed withsterile water, or a solution of Sodium hypochlorite (ranging from 0 ppmto ˜30,000 ppm active chlorine, including 50 ppm and 200 ppm activechlorine) for 3 to 20 minutes. Liquid was then drained. This processraises the internal moisture content to approximately 16%. Followingthis brief surface sanitation step, the seed internal moisture contentis lowered in a commercial seed dryer with a flow of dehumidified air(temperature controlled to approximately 60 to 90 degrees F.) to lessthan 8% (4 to 6% generally preferred). This drying step maintains seedvigor, yet loosens the papery seed hull (seed coat) for processing ease.This lowered moisture content seed is also significantly more brittle.This brittleness is employed to properly split the seed in the dehullingmill, thus maximizing recovery of high quality explants with vigorous,intact meristems. Seed thus prepared can be stored for a significantperiod of time (2 years or more under proper conditions), or be useddirectly for further processing.

2) Properly prepared dry soybean seeds can be split, and viable meristemexplants recovered using a variety of machines. One machine successfulemployed is a Grainman Rice Sheller (Model 64). Seeds split in thismachine can then be further processed to recover the desiredmeristem-bearing explants in a Clipper-Cleaner modified with the propersized screens. Explants recovered in this fast and gently process can bedirectly used for transformation, or can be stored until needed. Typicaltemperature conditions during storage can range from about roomtemperature to ⁻80 degrees C.

3) Following desired storage, explants were rehydrated fortransformation. The types of media used for this step can be varied andincluded “bean germination medium” (BGM; Media Table 16), soy inoculummedium (INO; Table 1), and prepared log-phase Agrobacterium growthcultures (AGRO). The Agrobacterium growth culture was grown overnight inLysogeny Broth (LB, also commonly referred to as Luria-Bertani Broth) tolog phase, and then centrifuged and resuspended to a final opticaldensity at 660 nm of 0.25 to 0.6. The medium used for the dilution isthe same as the soy inoculum medium. Rehydration temperatures anddurations also can be varied, with some experiments having explants thatwere soaked in one of these solutions overnight at 4° C. Othervariations were made in the duration of exposure to respective hydrationmedia, the various temperatures during this exposure, and the extent ofsaturation in the respective media. Exposure times tested ranged from 0to 24 hours. Temperatures during longer exposure times (those greaterthan 4 hours) were done at either room temp (˜26° C.), 23° C., or 4° C.Exposure times of 4 hours or less were all tested at room temperature.As an alternative to completely submerging or substantially saturatingexplants with liquid media during the hydration process, some treatmentsemployed the use of moistened filter paper (enough liquid to wet, butnot to saturate). This was done with filter paper moistened with eitherBGM or Agrobacterium-culture medium. Rehydration was performed in avariety of vessels, including but not limited to conical centrifugetubes, graduated glass bottles, or a PLANTCON tissue culture container(MP Biomedicals, Irvine, Calif.).

After rehydration, explants were briefly sonicated in the presence ofthe appropriate Agrobacterium cultures as described in other examples.Co-culture was performed in lighted Percivals for generally 2 to 5 days(16 hours of light, 8 hours of dark, light intensity ≧5 μE) at atemperature of approximately 23 to 25° C. Spectinomycin was applied as aselection agent either during rehydration, in co-culture steps, and/orfollowing co-culture at 15 mg/L to 1000 mg/L. Phenotype positive shoots(plants) were routinely recovered (see Table 17).

TABLE 16 Media for soybean germination. Ingredients of BGM mg/L NH₄NO₃240 KNO₃ 505 CaCl₂•2H₂O 176 MgSO₄•7H₂O 493 KH₂PO₄ 27 H₃BO₃ 1.86Na₂MoO₄•2H₂O 0.216 MnSO₄•H₂O 5.07 ZnSO₄•7H₂O 2.58 FeSO₄•7H₂O 2.502 KI0.249 Na₂EDTA •2H₂O 3.348 CuSO₄•5H₂O 0.0008 CoCl₂•6H₂O 0.0008 ThiamineHCl 1.34 Nicotinic Acid 0.5 Pyridoxine HCl 0.82 Bravo (75% WP) 30 Captan(50% WP) 30 Cefotaxime 125 Sucrose 25000 pH 5.8

As shown in Table 17, a transformation frequency of 20-25% was obtainedin several experiments, and over 10% was obtained routinely, dependingon the protocol used.

TABLE 17 Transformation frequency (TF) of dry-excised soybean explants.Experiment- treatment number # Explants # Events TF % 1095 138 5 3.61109 455 23 5.1 1141-1 543 136 25 1141-2 541 67 12.4 1169-1 192 37 19.31260 281 57 20.3 1261 770 154 20 1263-1 235 14 6 1262-2 59 11 18.61263-2 159 21 13.2 1264-1 55 9 16.4 1265-1 636 151 23.7 1264-2 102 4 3.91265-2 101 13 12.9

Example 6 Cotton Transformation Using aadA as a Selectable Marker andSpectinomycin as a Selective Agent

A. Preparation of Agrobacterium Inoculum

Agrobacterium strain C58 harboring a binary vector which carries 1 T or2 T-DNA containing aadA and other GOI or screenable marker was used. Theinoculum was prepared as described in Example 1.

B. Cotton Explants, Inoculation and Co-Culture with Agrobacterium.

Cotton embryo axes were mechanically excised from imbibed mature seedsand inoculation and co-cultured with prepared Agrobacterium wasperformed. Cotton seeds were mechanically processed to excise andisolate their meristematic tissues. In order to obtain transformablemeristematic explant material, cotton seeds (e.g. from genotypes STN474(Stoneville Pedigreed Seed Co., Stoneville, Miss.), Delta Pearl (Deltaand Pine Land Co., Scott, Miss.), DP5415 (Delta and Pine Land Co.),SureGrow501 or SureGrow747 (Sure Grow Cotton Seed Company, Maricopa,Ariz.) were processed as follows to separate the embryo, comprisingmeristematic tissues, from the seed coat and cotyledon(s). Cotton seedswere removed from storage at 4° C. or −20° C. and brought to roomtemperature. Seeds were weighed out, placed into a sterile germinatorunit, and surface-sterilized in 50% Clorox (sodium hypochlorite) for 5min. Seeds are then rinsed 3 times with sterile distilled water and werehydrated in a liquid hydration medium (CSM) at 28° C. in the dark forabout 18 hrs (range of 14 to 42 hours). Alternatively, the germinationtemperature may be lower, for instance about 23° C. The CSM mediumcontained 200 mg/L carbenicillin (PhytoTechnology Laboratories, ShawneeMission, Kans.), 125 mg/L cefotaxime (Midwest Scientific, St. Louis,Mo.), 30 mg/L BRAVO 75 (Carlin, Milwaukee, Wis.) and 30 mg/L Captan 50(Carlin). Other solutions have also successfully been used to hydratethe cotton seeds, including sterile deionized water or water containinga weak concentration of bleach (typically 50 to 1000 ppm sodiumhypochlorite). Following hydration, seeds may be used immediately, orstored at refrigeration temperatures for up to a week prior to furtherprocessing.

Explants were rinsed in sterile water. About 1-60 g, e.g. 30 g, ofexplants was placed into the top part (upside down) of a Plantcon™container (MP Biomedicals, Solon, Ohio) followed by addition ofapproximately 50 mL of the prepared Agrobacterium suspension, enough tocover the explants. After the Plantcon™ was closed, it was inserted intoan appropriately sized holder, which was placed into a sonicator (e.g.L&R Ultrasonics QS140; L&R Manufacturing Co., Kearny, N.J.; or a HondaW113 sonicator, Honda, Denshi Japan). The sonicator was filled withabout 2 L of 0.1% Triton® (e.g. Sigma 526-36-23; Sigma Chemical Co, St.Louis, Mo.). After up to 5 min of sonication, the Plantcon™ was placedsecurely on a shaker at about 80-100 rpm for 10 min for incubation.After inoculation, the Agrobacterium inoculum was removed from thePlantcon™. About 2 g of the inoculated explant tissue was transferred toa fresh Plantcon™ containing sterile filter paper and 5 mL of INO (Table1), and the explants were spread on the medium surface to avoidclustering. The INO medium may also be supplemented with plant growthregulators such as gibberellins (GA3), auxins (NAA, IBA, IAA, 2,4-D,dicamba, etc), cytokinins (BAP, thidiazuron, dikegulac, kinetin, etc.),and the antimicrobial compounds 50 ppm Nystatin (50 mg/L), TBZ (10mg/L), and the selection agent Spectinomycin (100 mg/L). The Plantcon™containing inoculated explants was placed into a Percival incubator forco-cultivation at approximately 22-28° C. and a 16 hour lightphotoperiod for 2-5 days (light intensity≧5 μE).

C. Selection and Identification of Transgenic Events Using Spectinomycinas a Selective Agent.

Following co-cultivation, explants were transferred onto semi-solidselection medium in Plantcon™ containers by either individuallyimplanting into the medium, or they were laid on the surface of themedium. The basal medium was a modified Lloyd & McCown Woody PlantMedium (WPM, Lloyd and McCown, 1981) and was supplemented with 200 mg/Lcefotaxime, 200 mg/L carbenicillin and 100-200 mg/L spectinomycin (Table18) with or without plant growth regulators or other additives topromote multiple shoot formation and growth.

TABLE 18 Components of medium for selection and shoot development usedin cotton transformation - Modified Lloyd & McCown Woody Plant Mediumsupplemented with antibiotics. Ingredient Amount/L LM WPM with vitamins(Phytotech L449) 2.41 g Dextrose (Fisher D16-3) 20 g Calcium gluconate(Sigma G-4625) 1.29 g With or without Clearys 3336 WP (Carlin 10-032)0.03 g AGARGEL (Sigma A-3301) 4 g Fill water to 1 L pH 5.6 AutoclaveCarbenicillin (Phytotech C346) (40 mg/mL stock) 5 mL (200 mg) Cefotaxime(Midwest NDC0039-0019-10) 4 mL (200 mg) (50 mg/mL stock) Spectinomycin(50 mg/mL stock) 3 mL (150 mg)

Twenty five to 50 explants were cultured in each container. The explantswere either immediately moved into light culture room (16-h light/8-hdark photoperiod, light intensity≧5 μE) with temperature set atapproximately 28° C., or first into light room with temperature set atapproximately 35° C., up to 40° C., for a short period of time (e.g. 3-5days) before being moved to 28° C. Experiments comparing these twoculture regimes were conducted and results suggested that treatment at35° C. was beneficial (Table 19).

TABLE 19 Comparison of inoculation and co-culture methods for cottontransformation using spectinomycin selection. Inoc/ co- # GUS+ shoots %explants cultivation Culture # explants w/ (total # shoots producingGUS+ Exp-Trt method temperature meristem assayed) shoots 1021-1 A 35°C., 3 d to 28° C. 127 6 (7) 4.7 1021-2 B 28° C. 183 0 (2) 0 1021-3 B 35°C., 3 d to 28° C. 141 0 (2) 0 1021-4 A 28° C. 324 2 (2) 0.6 1021-5 A 35°C., 3 d to 28° C. 225 7 (9) 3.1 1023-1 A 35° C., 3 d to 28° C. 81 5 (7)6.2 1023-2 B 28° C. 95 0 (0) 0 1023-3 B 35° C., 3 d to 28° C. 81 11 (13)13.6 1023-4 A 28° C. 101 0 (0) 0 1023-5 A 35° C., 3 d to 28° C. 88 1 (3)1.1

In method A, all explants in each treatment were placed in one PLANTCONand Agrobacterium inoculum was added to cover the explants. The explantsin the inoculum were sonicated (bulk sonication) to create wounds forAgrobacterium entry, for 2 min, followed by 10 min on shaker (80 rpm).Then the inoculum was removed and the explants were distributed toPLANTCONs each containing one piece of filter paper and 5 ml ofinoculation medium. In method B, explants were distributed to the coverpart of each PLANTCON (approximately 100 explants per PLANTCON). Five mlof Agrobacterium inoculum was added, and the explants were thensonicated for 20 sec, and immediately were transferred along with theinoculum to the bottom part of a PLANTCON, which holds one piece offilter paper.

After approximately 3-4 weeks on the selection medium, resistant greenshoots began to be evident on some explants, while bleached young shootsor primordia were clearly visible on others. In approximately another 2weeks on selection medium, those explants developing green shoots weretransferred to Oasis® plugs for shoot growth and root induction from theoriginal radical in the greenhouse. The Oasis plugs were placed in astandard flat without holes and were situated in a simple liquid medium,which contained 0.5 g/L of WPM salts with vitamins (PhytotechnologyLaboratories, Lenexa Kans.; stock No. L449) and 0.25 mg/L IBA, and werecovered with plastic domes. Explants might also be transferred ontofresh selection medium with the same or higher concentration ofspectinomycin, or with spectinomycin removed, for further selectionand/or growth before being moved to the plugs. In some experiments,cotton explants were subjected to tissue culture and growth conditionsessentially as described for soybean transformation, selection, andplant regeneration of Protocol “D”, above. Cotton rooting medium (CRM;Table 20) might also be used to induce formation of roots.

TABLE 20 Components of Cotton Rooting Medium (CRM). Ingredient Amount/LMS basal salts (Phytotech M524) 2.15 g Myo-inositol (Sigma I-3011) 0.1 gDextrose (Fisher D16-3) 30 g SBRM vitamin stock: 2 mL Glycine (SigmaG-6143): 1 g/L Nicotinic acid (Sigma N-0765): 0.25 g/L Pyridoxine HCl(Sigma P-8666): 0.25 g/L Thiamine HCl (Sigma T-3902): 0.5 g/L Cysteine(10 mg/mL) 10 mL Bring volume with deionized distilled H₂O pH with KOH5.8 Bacto agar (BD 214030) 8 g Autoclave IAA (Sigma I-2886) (0.02 mg/mL)5 mL Timentin (Duchefa T0190) (100 mg/mL) 1 mL Cefotaxime (MidwestNDC0039-0019-10) (50 mg/mL) 4 mL

In approximately 3-4 weeks, most of the shoots in Oasis® plugs had grownsignificantly and roots were also well developed. Tissues were assayedfor molecular characterization by one or more molecular assay methods,e.g. Invader® assay (Third Wave™ Technologies, Madison, Wis.), PCR, orSouthern hybridization. Leaf samples could also be collected from eachgreen shoot and assayed for GUS activity while still on the selectionmedium and/or at later stage, if a construct containing a uidA gene wereused. Spectinomycin served as a useful visual marker for earlyidentification of transformation. Non-transformed tissues usuallyappeared bleached and often malformed under spectinomycin selection,whereas transformed tissues were green and properly developing. Inexperiments utilizing a uidA marker gene, the transformed nature of thegreen tissue could be confirmed by GUS expression after about 4-8 weekson selection media. Therefore, using spectinomycin as a selection agentforegoes the labor intensive and time consuming GUS assays often used inmeristem transformation systems, and provides the advantage ofsignificantly reducing the labor involved in producing transgenicplants.

Example 7 Corn Transformation Using aadA as a Selectable Marker Gene

A. Corn Explants

Ears containing immature embryos (e.g. FBLL or LH244) are harvestedapproximately 10 days after pollination and kept refrigerated at 4° C.until use (up to 5 days post-harvest). The preferred embryo size forthis method of transformation is ˜1.0-2.0 mm. This size is usuallyachieved about 10 days after pollination inside the greenhouse withgrowth conditions of an average temperature of 87° F., day length of 14hours with supplemental lighting supplied by GE 1000 Watt High PressureSodium lamps. The method is genotype independent.

B. Preparation of Agrobacterium Inoculum

Agrobacterium strain C58 harboring a binary vector which carries 1 T- or2 T-DNA containing aadA and other GOI or screenable marker are used. Theinoculum can be prepared as described in US Patent ApplicationPublication No. 20040244075.

C. Inoculation and Co-Culture

Immature embryos are isolated from surface sterilized ears and directlydropped into the prepared Agrobacterium cell suspension in 1.5-mLmicrocentrifuge tube. The isolation lasts continuously for 15 min. Thetube is then set aside for 5 min, which makes the inoculation time forindividual embryos range from 5 to 20 min. After the Agrobacterium cellsuspension is removed using a fine tipped sterile transfer pipette, theimmature embryos are transferred onto co-culture medium (Table 21). Theembryos are placed on the medium with the scutellum side facing up. Theembryos are cultured in a dark incubator (23° C.) for approximately 24h.

D. Selection, Regeneration and Growth of Transformants onSpectinomycin-Containing Medium

After the co-cultivation, the embryos are transferred onto a modified MSmedium (Induction MS, Table 21) supplemented with 500 mg/L carbenicillinand 50, 100, 150, 200, or 500 mg/L mg/L spectinomycin in Petri dishes(100 mm×25 mm), 20 to 25 embryos per plate. Auxin and cytokinin arepresent to initiate an embryogenic culture response from the scutellartissue. The plates are kept in a dark culture room at 27° C. forapproximately 2 weeks. Immature embryos with callus developed aretransferred individually onto the first regeneration medium, the samemedium mentioned above except 2,4-D and picloram are replaced by 3.5mg/L BAP (MS/BAP, Table 21) and the carbenicillin level is reduced to250 mg/L. The cultures are moved to a culture room with 16-h light/8-hdark photoperiod and 27° C. After 5-7 days, the callus pieces may alsobe transferred onto the second regeneration medium, a hormone-freeMS-based medium (MSOD, Table 21) in Petri dishes (100 mm×25 mm). Afterapproximately another 2 weeks, the callus pieces that have green shootsregenerated or are still alive are transferred onto the samehormone-free medium in Phytatrays for further selection and growth. Allmedia mentioned above is supplemented with 50, 100, 150 or 200 mg/Lspectinomycin. Regenerated green plants (R0) are moved to soil in peatpots in a growth chamber when they reach the top of Phytatrays and haveone or more healthy roots. After an additional 7 to 10 days, they arethen transplanted into 12-in pots and moved to greenhouse withconditions for normal corn plant growth. The plants are eitherself-pollinated or crossed with wild-type plants.

Molecular assays (e.g. as described above for cotton plants) areconducted to characterize the plants.

TABLE 21 Culture media for use in transforming and regenerating corn. ½MS Co-culture Induction Component ½ MS VI PL medium MS MSW50 MS/6BA MSODMS salts 68.5 g/l 68.5 g/l  2.2 g/l  4.4 g/l  4.4 g/l  4.4 g/l  4.4 g/lSucrose   20 g/l 68.6 g/l  20 g/l   30 g/l   30 g/l   30 g/l — Maltose —— — — — —   20 g/l Glucose   10 g/l   36 g/l  10 g/l — — —   10 g/l1-Proline  115 mg/l  115 mg/l 115 mg/l 1.36 g/l 1.38 g/l 1.36 g/l —Casamino Acids — — —   50 mg/l  500 mg/l   50 mg/l — Glycine   2 mg/l  2 mg/l  2 mg/l —   2 mg/l — — l-Asparagine — — — — — —  150 mg/lmyo-Inositol  100 mg/l  100 mg/l 100 mg/l —  100 mg/l —  100 mg/lNicotinic Acid  0.5 mg/l  0.5 mg/l  0.5 mg/l  1.3 mg/l  0.5 mg/l  1.3mg/l  1.3 mg/l Pyridoxine•HCl  0.5 mg/l  0.5 mg/l  0.5 mg/l 0.25 mg/l 0.5 mg/l 0.25 mg/l 0.25 mg/l Thiamine•HCl  0.1 mg/l  0.1 mg/l  0.6 mg/l0.25 mg/l  0.6 mg/l 0.25 mg/l 0.25 mg/l Ca Pantothenate — — — 0.25 mg/l— 0.25 mg/l 0.25 mg/l 2,4-D — —  3 mg/l  0.5 mg/l  0.5 mg/l — — Picloram— — —  2.2 mg/l — — — Silver Nitrate — —  1.7 mg/l  1.7 mg/l — — — BAP —— — — —  3.5 mg/l — ¹Media ½ MSVI and ½ MSPL are used as liquid.Co-culture medium is solidified with 5.5 mg/l low EEO agarose. All othermedia are solidified with 7 g/l Phytagar or 3 g/l phytagel forglyphosate selection.

Example 8 Preparation of an Enhanced 16S RNA Promoter from Agrobacteriumand CTP-aadA Fusion Genes

pMON107379, a 2 T-DNA vector with OriRi replication origin has apromoter located in the backbone (i.e. outside of the T-DNA) forselection of spectinomycin resistance in E. coli or Agrobacterium hostcells. To make pMON107379, the plant spec selection cassette was excisedfrom pMON96999 (FIG. 2) with NotI digestion and inserted into pMON107341opened with PspOMI. The parental pMON107341 is an oriRi based vectorwith an improved spectinomycin resistance cassette driven by P-rrnpromoter. An enhanced 16S RNA promoter from Agrobacterium (SEQ ID NO:3)is especially useful when the copy number of the vector is low, as withvectors containing OriRi. The P-rrn promoter was isolated from theAgrobacterium strain C58 16S rDNA by PCR, and fused to the virE operonribosomal binding site (RBS) to enable its efficient translation in bothE. coli and Agrobacterium. pMON107379 also comprises an aadA gene thatencodes an aminoglycoside-3′-adenyltransferase (SEQ ID NO:1) conferringspectinomycin resistance, the gene encoding anaminoglycoside-3′-adenyltransferase also being fused with a chloroplasttransit peptide (SEQ ID NO:2) for transport of the nuclear-encodedaminoglycoside-3′-adenyltransferase to plastids.

Example 9 Direct Retransformation of Elite ROUNDUP READY™ Germplasm inSoybean and Cotton

Utilizing methods described herein, elite transgenic Round-Up Ready™germplasm can be transformed utilizing 2 T-DNA's encoding the aadA genefor spectinomycin selection while employing a new gene (often referredas “the gene of interest”) on the second T-DNA (or plasmid, if twoplasmids are used) to allow segregation away from the aadA as described.

A Cotton RRFlex® seed variety (07W610F) and germplasm controlnon-transgenic variety (00S04) were compared. Seed was imbibed for ˜18hrs in 24° C., machine excised and machine-sieved (in two steps)following by floatation enrichment of explants. Explants were inoculatedwith Agrobacterium suspension in INO at OD₆₆₀ 0.3, sonicated for 2 min,and incubated for 10 min. The Agrobacterium suspension was then removedand explants distributed into co-culture containers at approximately 2 gper container. Explants were laid onto filter papers wetted with 5 ml ofco-culture media (INO with additions of 50 ppm Nystatin, 10 ppm TBZ, and100 ppm Spectinomycin) and co-cultured in a lighted Percival incubatorat approximately 23 to 25° C. (16 hrs light/8 hrs dark, lightintensity≧5 μE) for 3 days. Explants were then transferred onto WPMmedia with 150 ppm spectinomycin, incubated for 3 days in 35° C. lightroom (16 hrs light/8 hrs dark), and then moved to 28° C. light room (16hrs light/8 hrs dark). Phenotype positive green plantlets were harvested6 weeks after inoculation, placed in Oasis® plugs (Smithers-Oasis USA;Kent, Ohio) wetted with 0.5 g/L WPM salts (optionally including IBA at0.25 mg/L to improve rooting) and moved to green house conditions. Onceplants acclimatized and started to grow they were assayed for CP4, GUS,and vascular GUS expression (a predictor of germline transformation).Retransformed transgenic plants were expected to be CP4+ GUS+, whiletransformed control plants were expected to be CP4− GUS+. An analysis ofthe yield of transformed plants is listed in Table 22 below. Totaltransformation frequency is expected to increase, as analysis is notcomplete at this time. The described procedure is useful to re-transformtransgenic cotton plants with an efficiency similar to transformation ofa conventional non-transgenic cotton variety.

TABLE 22 Transformation frequency of retransformed germplasm. PlantsSpectinomycin Number of expressing % TF, Quality phenotype plants GUS inall germline Cotton explants positive (green) % green sampled forleaves, expressing Germplasm inoculated plantlets Plantlets GUS(germline) GUS 00S04 928 18 1.90% 11 2 0.22% 07W610F 3665 87 2.40% 64 90.25%

Example 10 Sterilization of Seeds and/or Explant Material

A number of techniques of sterilizing seeds before excision, as well assterilizing explants after excision from the seeds were tested.Post-excision sterilization of dry explants using chlorine gas in avacuum desiccation chamber was tested at time intervals ranging from 15minutes to 16 hours. Contamination control increased with longerexposure to Cl gas, although fungal contamination grew in treatments inwhich the exposure to Cl gas had surpassed the survivable threshold ofthe explants.

Ozone gas treatments were also tested. Both whole seed (before excision)and dry explants (after excision) were exposed to O₃ gas in a PLEXIGLASchamber (OSR-8 Ozone Generator; Ozone Solutions, Sioux Center, Iowa) atvarious time intervals of 1-24 hours. O₃ was used at a concentration of467 ppm. After seed was exposed to ozone, embryonic material was excisedand explant viability was measured. Ozonation of soybean seed for 12hours or less did not impact viability of subsequently isolatedexplants, but drastically decreased bioburden found in explants.Ozonation of dry excised explants for as little as 1-4 hours decreasedexplant health (i.e. number of viable embryos).

Additional tests on pre-excision sterilization of whole seed wereperformed using a bleach solution of 200 ppm active chlorine, followedby an overnight hydration period (˜9 hours) in a solution of 50 ppmactive chlorine. These seeds were then allowed to dry in a laminar flowhood (typically for 12-48 hours) before being excised mechanically. Amodification to the 50% bleach soak protocol was also tested, in whichthe seeds were first rinsed with a 70% solution of ethanol. The ethanolwas immediately drained (total exposure to ethanol was less than 5seconds), and then the 50% bleach soak was performed by treating seeds3-15 min in 50% bleach followed by 3 rinses with water and drying theseeds overnight such that the moisture content was less than 8%. UVlight may also be employed to sterilize the plant material.

Example 11 Hydration of Seeds and Explant Material

Studies employing new pre-culture hydration/germination strategies weretested. The types of media used for this step included “bean germinationmedium” (BGM; Table 16), soy inoculum medium (INO; Table 1), andprepared log-phase Agrobacterium growth cultures (AGRO). TheAgrobacterium growth culture was grown overnight in Lysogeny Broth (LB,also commonly referred to as Luria-Bertani Broth) to log phase, and thencentrifuged and resuspended to a final optical density at 660 nm of 0.25to 0.6. The medium used for the dilution is the same as the soy inoculummedium. Explants were soaked in this solution overnight at 4° C. Othervariations were made in the duration of exposure to respective hydrationmedia, the various temperatures during this exposure, and the extent ofsaturation in the respective media. Exposure times tested ranged from 0to 24 hours. Temperatures during longer exposure times (those greaterthan 4 hours) were either room temp (˜26° C.), 23° C., or 4° C. Exposuretimes of 4 hours or less were all tested at room temperature. As analternative to completely submerging or substantially saturatingexplants with liquid media during the hydration process, some treatmentsemployed the use of moistened filter paper (enough liquid to wet, butnot to saturate). This was done with filter paper moistened with eitherBGM or Agrobacterium-culture medium. Rehydration was performed in avariety of vessels, including but not limited to conical centrifugetubes, graduated glass bottles, or a PLANTCON tissue culture container(MP Biomedicals, Irvine, Calif.).

This example also demonstrates that hydration can be done in a varietyof media containing various types of carbohydrates such as glucose(INO), and sucrose (BGM). Other carbohydrates such as galactose may beuseful in hydration medium.

Example 12 Transformation of Dry-Excised Soy Explants Stored forExtended Periods of Time

Dry-excised explants were stored for up to 20 months at −20° C. to 4°C., and then tested for survival, transformability and vigor. Explantsurvival and overall vigor appeared to be similar in all treatmentgroups, regardless of storage conditions or temperature compared tocontrol treatment (Treatment 1). This demonstrates the ability to storedry-excised explants for almost two years without detriment. Explantsfrom each treatment were tested for transient GUS expression 4 daysafter inoculation. Table 23 shows a comparison of meristem specific gusexpression between treatments, scored on a scale from 0-9, with 0 beingno visible expression, and 9 being extensive expression in all 3meristems of the embryo. This demonstrates that dry-excised explants cannot only survive long-term storage in various conditions withoutsignificant loss of vigor, but they also retain amenability totransformation. Thus it is now possible to excise large quantities ofexplants during off-peak times for later use, which representssignificant potential cost savings and flexibility in planning andexecuting transformation studies.

TABLE 23 Effect of storage duration and temperature on explanttransformation. Seed Transient gus Sterilization Excision StorageStorage expression Treatment Technique technique duration temperature(scale of 0-9) 1 & 2 50% bleach Automated dry None NA 0.90, 1.60 rinseexcision with Grainman Rice dehuller 3 50% bleach Automated dry 17months    4° C. 0.20 rinse excision with Grainman Rice dehuller 4 50%bleach Automated dry 17 months −20° C. 0.10 rinse excision with GrainmanRice dehuller 5 50% bleach Manual dry 20 months    4° C. 0.70 rinseexcision 6 50% bleach Manual dry 20 months −20° C. 1.50 rinse excision

Example 13 Identification of Suitable Pre-Inoculation Culture(“Pre-Culture”) Compositions and Conditions

It is likely that dry excised explants are still in a state ofsemi-dormancy when they are inoculated with Agrobacterium fortransformation. Thus a method was developed to stimulate the metabolicactivity of the dry excised explants prior to Agrobacterium inoculation,for increasing their transformation competency. That is, by manipulatingthe biology of the dry explant, it is possible to increase % germlinepositive events per explant by 2 to 10 fold.

Several media compositions: BGM (Table 16), INO (Table 1), or OR (Table24) were tested at 23° C. and/or 28° C. temperatures, and underdifferent light/dark conditions from 1 to 5 days, for their ability toenhance transformation competency. After pre-culturing step, explantswere pooled together and inoculated with the Agrobacterium cultureaccording to the method described in Example 1. Transient GUS expressionassays performed on explants showed increased GUS activity in thepre-cultured treatments after 2 days and 4 days of co-culture.

Plant losses occurred due to fungal infection in some of thepre-culturing experiments, but overall TF of the dry excised explantsthat were pre-cultured on filter papers wetted with BGM at 23° C. indark for 5 days appeared to be highest when compared with dry excisedexplants that were not pre-cultured. The losses due to fungalcontamination could be mitigated by using an anti-fungal agent such asBRAVO 75 and Captan 50 at about 1% each during the pre-culture and/orco-culture step. Southern blot and INVADER analysis of the plantsproduced in this example with a CP4 probe confirmed the transgenicnature of these plants.

TABLE 24 SOY Organogenic (OR) MEDIUM COMPOUND: PER 4 LITER: MS Salts17.2 g 3X Minor MS Salts 40 ml Nicotinic Acid (1 mg/ml) 4 ml PyridoxineHCl (1 mg/ml) 4 ml Thiamine HCl (1 mg/ml) 46.8 ml Sucrose (Ultra Pure)120 g Myo-Inositol (Cell Culture Grade) .40 g pH 5.8 Washed Agar 32 gADDITIONS AFTER AUTOCLAVING: Proline (2.5 m Stock) 19.2 ml TSG/ORHormone Stock 40.0 ml

TABLE 25 Effect of pre-culture of dry explant; transformation frequencyusing pMON10343. Pre-culture Media compositions and Rooted % Fungal lossExplant Type conditions Explants Shoots TF (PLANTCONs) WET None 300 155.00% 0% DRY None 650 6 0.92% 13% DRY BGM, 5 d 23 C. dark 972 29 2.98%0% DRY BGM, 5 d 23 C. 16/8 365 1 0.27% 44% light DRY BGM, 5 d 28 C. dark315 3 0.95% 7% DRY BGM, 5 d 28 C. 16/8 188 1 0.53% 62% light

Studies were repeated comparing two constructs, pMON101343, comprisingone T-DNA that comprises a CP4 gene specifying glyphosate resistance andan OriV replication origin; and pMON107350 comprising one T-DNA thatcomprises a CP4 gene specifying glyphosate resistance and an OriRreplication origin (US20070074314) in the vector backbone. Again,pre-culturing of dry explants boosted TF as compared to the TF of nonpre-cultured dry explants, as shown in Table 26.

TABLE 26 Additional studies on pre-culture of dry-excised explants.Explant type and vector # Explants # Rooted Shoots TF pMON101343 WET 53516 2.99% DRY 1331 8 0.60% DRY PRECULTURE 2437 43 1.76% pMON107350 WET671 11 1.64% DRY 190 0 0.00% DRY PRE-CULTURE 500 9 1.80%

As shown in Table 27 pre-cultured dry excised explants also yieldedhigher TFs when explants were cultured in liquid regeneration medium(media of Table 12 except for AgarGel) which was removed and addedautomatically using a robotic system. TF appeared to be even higher withthe liquid regeneration medium with a pre-culturing step. Wet excisedexplants in liquid media appear to have had low TF due to contamination.

Pre-culturing surprisingly improves competency for transformation andimproves transformation uniformity. Such improvements are crucial toreducing variability during production runs at industrial scale forproducing transgenic soybean plants.

TABLE 27 Pre-culture of dry excised explants; comparison of solid andliquid media. Pre-culture Explant type Media compositions RegenerationRooted pMON101343 and conditions medium Explants Shoots TF WET Nonesolid WPM 460 17 3.70% WET None liquid WPM 31 0 0.00% DRY None solid WPM1286 8 0.62% DRY None liquid WPM 128 0 0.00% DRY BGM, 5 d 23 C. darksolid WPM 1257 33 2.63% DRY BGM, 5 d 23 C. dark liquid WPM 111 3 2.70%

Example 14 Production of Transgenic Soybean Plants Using Dry SoybeanExplants and Spectinomycin Selection

Dry, viable, seeds (properly stored quality soybean seed compriseapproximately 10 to 12% internal moisture content) were rinsed withsterile water, or a solution of Sodium hypochlorite (ranging from 0 ppmto ˜30,000 ppm active chlorine, including 50 ppm and 200 ppm activechlorine) for 3 to 20 minutes. Liquid was then drained. This processraises the internal moisture content to approximately 16%. Followingthis brief surface sanitation step, the seed internal moisture contentwas lowered in a commercial seed dryer with a flow of dehumidified air(temperature controlled to approximately 60 to 90 degrees F.) to lessthan 8%.

Following desired storage, explants were rehydrated for transformation.The types of media used for this step may be varied and included “beangermination medium” (BGM; Table 16), soy inoculum medium (INO; Table 1),and prepared log-phase Agrobacterium growth cultures (AGRO). TheAgrobacterium growth culture was grown overnight in Lysogeny Broth (LB,also commonly referred to as Luria-Bertani Broth) to log phase, and thencentrifuged and resuspended to a final optical density at 660 nm of 0.25to 0.6. The medium used for the dilution is the same as the soy inoculummedium. Rehydration temperatures and durations also can be varied, withsome experiments having explants that were soaked in one of thesesolutions overnight at 4° C. Other variations were made in the durationof exposure to respective hydration media, the various temperaturesduring this exposure, and the extent of saturation in the respectivemedia. Exposure times tested ranged from 0 to 24 hours. Temperaturesduring longer exposure times (those greater than 4 hours) were done ateither room temp (˜26° C.), 23° C., or 4° C. Exposure times of 4 hoursor less were all tested at room temperature. As an alternative tocompletely submerging or substantially saturating explants with liquidmedia during the hydration process, some treatments employed the use ofmoistened filter paper (enough liquid to wet, but not to saturate). Thiswas done with filter paper moistened with either BGM orAgrobacterium-culture medium. Rehydration was performed in a variety ofvessels, including but not limited to conical centrifuge tubes,graduated glass bottles, or a PLANTCON tissue culture container (MPBiomedicals, Irvine, Calif.).

After rehydration, explants were briefly sonicated in the presence ofthe appropriate Agrobacterium cultures. Co-culture and subsequent stepswere performed in lighted Percival incubators for 2 to 5 days (16 hoursof light, 8 hours of dark, with light intensity of about 5 μE to 200 μE)at a temperature of approximately 23 to 25° C., and may be performed upto about 35° C. Light is known to promote gene transfer fromAgrobacterium to plant cells. Spectinomycin was applied as a selectionagent either during rehydration, in co-culture steps, and/or followingco-culture at 15 mg/L to 1000 mg/L.

Phenotype positive shoots (plants) were routinely recovered, as shown inTable 28, using the construct, pMON96999, comprising one T-DNAcomprising an aadA gene and an OriV origin of replication or theconstruct, or pMON101343 comprising one T-DNA comprising a CP4 gene andan OriV origin of replication. By “phenotype positive” in the presenceof spectinomycin, it is meant that shoots are green and robust, whilephenotype negative shoots are weak and bleached (white), if theyelongate at all. Spectinomycin or glyphosate were used in theregeneration medium (both sold or liquid) at the concentration shown inTable 28.

TABLE 28 Transformation frequency of dry soybean explants usingglyphosate or spectinomycin as selective agent. Spectinomycin (% TF)Glyphosate (% TF) 25 ppm 50 ppm 100 ppm 200 ppm 50 uM 4.66 4.24 6.345.99 2.00

Spectinomycin was also used as a selective agent for transformation ofdry excised soybean embryos utilizing the following conditions: 1 hrhydration in INO medium, 4 days co-culture in INO, 150 ppmspectinomycin, with culture on solid or liquid WPM (Table 2; with orwithout added agar). Temperatures of 23-25 or 28° C., up to about 35°C., may be utilized. Phenotype positive shoots were harvested at 8 and10 weeks post Agrobacterium inoculation, and rooting was induced onsolid BRM (see Example 2) with 150 ppm Spectinomycin. Very hightransformation frequencies of 25.05% and 19.27% were obtained in twodifferent studies.

Example 15 Production of Transgenic Soybean Plants Using Dry SoybeanEmbryos, Spectinomycin, and Liquid Culture Medium

In these studies, explants were initially hydrated and eventuallyregenerated on WPM solid media with liquid overlay or WPM liquid mediumas above. All explants were transferred at 6 weeks post inoculation totrays containing Oasis® Wedge System (Smithers-Oasis USA; Kent, Ohio)and a simplified liquid medium (0.5 g/L WPM with 0.25 mg/L IBA). Rootedand shooted R₀ plants were obtained two to 4 weeks later. In all studiesand treatments, initial hydration of explants was done for 1 hour in therespective media as shown in the Table 29. Liquid culture medium was thesame as in Table 2 except glyphosate was replaced by spectinomycin at150 ppm. In liquid overlay treatment both solid and liquid culture mediawere used; liquid medium was dispensed over the top of explants as theywere lying on solid medium at a specified time during tissue culture asidentified in the Table 30. This was done as a type of media refreshmentand avoids the need for transferring explants from old media to newmedia. In the control treatments, explants were surface plated on asolid WPM medium (Table 2). Shoots were harvested and rooted on solidBRM as described above, except glyphosate was replaced withspectinomycin at 150 ppm.

TABLE 29 Transformation frequency with given hydration conditions. TF %Incubation with (mean of 3 Treatment Hydration medium Agrobacteriarepeats) 1-Control INO 0 minutes 3.10% 2 BGM w/o cefotaxime 0 minutes14.67% 3 BGM w/o cefotaxime 15 minutes 15.45% 4 BGM w/o cefotaxime 30minutes 18.50% 5 INO 0 minutes 13.98% 6 INO 15 minutes 9.64% 7 INO 30minutes 13.79%

TABLE 30 Liquid overlay timing. Oasis® Liquid medium Wedge TF % Liquidoverlay volume transfer for (mean of Treatment overlay timing on solidWPM regeneration repeats) Control- 1 NA None No 8.00% 2 None None Yes14.67% 3 3 weeks post  5 mLs Yes 15.45% inoculation 4 3 weeks post 10mLs Yes 18.50% inoculation 5 4 weeks post  5 mLs Yes 13.98% inoculation6 4 weeks post 10 mLs Yes 9.64% inoculation

Example 16 Production of Transgenic Soybean Plants Using Dry SoybeanEmbryos, Spectinomycin, and Transferring the Whole Regenerated Explantwith a Pre-Culturing Step

In these studies, as with Example 13, a pre-culturing step (5 days 23°C. dark in BGM) was used. A one hour hydration of the dry excisedexplant on INO medium was also done before the pre-culturing step. About12 mls of liquid WPM containing 150 ppm of spectinomycin was dispenseddirectly into the co-culture PLANTCON after the co-culture period, andexplants were surface plated on solid WPM containing 150 ppmspectinomycin 4 days later. In this example, phenotype positive greenshoots were identified at about week 4 of regeneration and transferredfrom WPM regeneration medium to trays containing Oasis® Wedge System(Smithers-Oasis USA; Kent, Ohio) and a simplified liquid medium (0.5 g/LWPM with 0.25 mg/L IBA). Rooted and shooted R₀ plants were obtained twoto 4 weeks later. Overall, pre-culturing in these studies also improvedTF % (Table 31). Percentage quality events shown below (Table 22) refersto the proportion of transgenic events demonstrating the presence of 1-2copies of both a gene of interest (GUS) and a marker gene (aadA) byInvader™ assay. Estimated marker-free TF (mTF) refers the % of eventswithout the marker gene.

TABLE 31 Transformation frequency and quality observed from wholeregenerated explants. # % Estimated Protocol & # # Events Events qualitymTF vector type Explants produced TF % assayed events qTF % %** DryExcised - 260 34 13.1 +/− 0.17 32 21.9 2.7 +/− 0.23 0.62 2T/OriV DryExcised - 161 15 9.32 +/− 7.38 14 28.6 2.5 0.45 2T/OriRi Pre-culturedDry - 1641 319 19.4 +/− 5.42 311 24.4 4.6 +/− 1.35 1.1 2T/OriVPre-cultured Dry - 336 66 19.64 +/− 1.97  64 20.3 3.9 +/− 1.22 0.72T/OriRi

Example 17 Production of Transgenic Soybean Plants Using Stored DrySoybean Embryos, Spectinomycin, and Transfer of Whole RegeneratedExplant with a Pre-Culturing Step

In this example, 3 months stored dry explants were used, and a 1 hrhydration step done in INO was utilized, on dry excised explant.Pre-culturing was performed for 5 days at 23° C. in dark conditions inBGM with 50 ppm nystatin and 10 ppm TBZ fungicides. TDZ and lipoic acidwere both added to the inoculum and to the co-culture media (INO). Theconstruct, pMON107379, was a conventional 2T vector comprising oriRi andaadA gene, and co-culture was done for 5 days. After co-culture theexplants were surface plated on solid WPM and then transferred to theOasis® Wedge System (Smithers-Oasis USA; Kent, Ohio) with a simplifiedliquid medium (0.5 g/L WPM with 0.25 mg/L IBA). As shown in Table 32,pre-culturing dry explants boosted TF. Thus, 3 month old stored dryexplants could perform similarly to freshly excised dry explants.Further, the addition to INO Co-culture media of nystatin (50 ppm) andthiabendazole (10 ppm) dissolved in DMSO (1.0 ml of DMSO per liter ofINO) improved the health of explants, likely by controlling yeasts andfungi commonly found in and on seeds and can be a useful tool whenperforming large and/or automated tissue culture.

TABLE 32 Effect of pre-culture on TF (%) of stored dry explants. Explanttype Pre-culture step # Explants R0 plants TF Wet Excised No 263 7528.52% Stored Dry Explants No 678 71 10.47% Fresh Dry Explants No 375 246.40% Stored Dry Explants Yes 901 129 14.32% Fresh Dry Explants Yes 1008112 11.11%

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of the foregoing illustrative embodiments, itwill be apparent to those of skill in the art that variations, changes,modifications, and alterations may be applied to the composition,methods, and in the steps or in the sequence of steps of the methodsdescribed herein, without departing from the true concept, spirit, andscope of the invention. More specifically, it will be apparent thatcertain agents that are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope, and concept of the invention as defined by theappended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

-   U.S. Pat. No. 4,761,373; U.S. Pat. No. 4,810,648; U.S. Pat. No.    5,013,659; U.S. Pat. No. 5,015,580; U.S. Pat. No. 5,073,675; U.S.    Pat. No. 5,094,945; U.S. Pat. No. 5,141,870; U.S. Pat. No.    5,164,310; U.S. Pat. No. 5,217,902; U.S. Pat. No. 5,229,114; U.S.    Pat. No. 5,273,894; U.S. Pat. No. 5,276,268; U.S. Pat. No.    5,322,938; U.S. Pat. No. 5,352,605; U.S. Pat. No. 5,359,142; U.S.    Pat. No. 5,362,865; U.S. Pat. No. 5,378,824; U.S. Pat. No.    5,463,175; U.S. Pat. No. 5,512,466; U.S. Pat. No. 5,512,466; U.S.    Pat. No. 5,538,880; U.S. Pat. No. 5,543,576; U.S. Pat. No.    5,550,318; U.S. Pat. No. 5,561,236; U.S. Pat. No. 5,563,055; U.S.    Pat. No. 5,591,616; U.S. Pat. No. 5,605,011; U.S. Pat. No.    5,608,149; U.S. Pat. No. 5,627,061; U.S. Pat. No. 5,633,435; U.S.    Pat. No. 5,633,437; U.S. Pat. No. 5,637,489; U.S. Pat. No.    5,646,024; U.S. Pat. No. 5,689,041; U.S. Pat. No. 5,693,512; U.S.    Pat. No. 5,731,179; U.S. Pat. No. 5,750,876; U.S. Pat. No.    5,767,366; U.S. Pat. No. 5,824,877; U.S. Pat. No. 5,850,019; U.S.    Pat. No. 5,869,720; U.S. Pat. No. 5,914,451; U.S. Pat. No.    5,958,745; U.S. Pat. No. 5,981,834; U.S. Pat. No. 5,981,840; U.S.    Pat. No. 5,985,605; U.S. Pat. No. 5,998,700; U.S. Pat. No.    6,011,199; U.S. Pat. No. 6,040,497; U.S. Pat. No. 6,072,103; U.S.    Pat. No. 6,080,560; U.S. Pat. No. 6,140,075; U.S. Pat. No.    6,166,292; U.S. Pat. No. 6,171,640; U.S. Pat. No. 6,225,105; U.S.    Pat. No. 6,228,623; U.S. Pat. No. 6,265,638; U.S. Pat. No.    6,271,443; U.S. Pat. No. 6,380,462; U.S. Pat. No. 6,380,466; U.S.    Pat. No. 6,384,301; U.S. Pat. No. 6,414,222; U.S. Pat. No.    6,426,447; U.S. Pat. No. 6,444,876; U.S. Pat. No. 6,459,018; U.S.    Pat. No. 6,476,295; U.S. Pat. No. 6,483,008; U.S. Pat. No.    6,489,461; U.S. Pat. No. 6,495,739; U.S. Pat. No. 6,531,648; U.S.    Pat. No. 6,537,750; U.S. Pat. No. 6,538,178; U.S. Pat. No.    6,538,179; U.S. Pat. No. 6,538,181; U.S. Pat. No. 6,541,259; U.S.    Pat. No. 6,576,818; U.S. Pat. No. 6,589,767; U.S. Pat. No.    6,596,538; U.S. Pat. No. 6,613,963; U.S. Pat. No. 6,653,530; U.S.    Pat. No. 6,660,849; U.S. Pat. No. 6,706,950; U.S. Pat. No.    6,723,837; U.S. Pat. No. 6,770,465; U.S. Pat. No. 6,774,283; U.S.    Pat. No. 6,812,379; U.S. Pat. No. 6,822,141; U.S. Pat. No.    7,022,896; U.S. Pat. No. 6,828,475; U.S. Pat. No. 5,106,739; U.S.    Pat. No. 5,378,619; U.S. Pat. No. 5,530,196; U.S. Pat. No.    5,641,876; U.S. Pat. No. 5,659,122; U.S. Pat. No. 5,837,848; U.S.    Pat. No. 6,051,753; U.S. Pat. No. 6,140,078; U.S. Pat. No.    6,175,060; U.S. Pat. No. 6,177,611; U.S. Pat. No. 6,232,526; U.S.    Pat. No. 6,252,138; U.S. Pat. No. 6,294,714; U.S. Pat. No.    6,426,446; U.S. Pat. No. 6,429,357; U.S. Pat. No. 6,429,362; U.S.    Pat. No. 6,433,252; U.S. Pat. No. 6,437,217; U.S. Pat. No.    6,635,806; U.S. Pat. No. 7,002,058; U.S. Pat. No. 7,288,694.-   U.S. Pat. RE37,543-   U.S. Patent Application Publication 2005/0005321; U.S. Patent    Application Publication 2006/0059589; U.S. Patent Application    Publication 2003/0028917; U.S. Patent Application Publication    2003/0083480; U.S. Patent Application Publication 2003/0115626; U.S.    Patent Application Publication 2003/0135879; U.S. Patent Application    Publication 2003/110532; U.S. Patent Application Publication    2004/0177399; US Patent Application Publication No. 2004/0244075;    U.S. Patent Application Publication 2005/0183170; U.S. Patent    Application Publication 2005/0022261; U.S. Patent Application    Publication 2006/0200878; U.S. Patent Application Publication    2007/0271627.-   Bevan et al., Nature, 304:184-187, 1983-   Broothaerts et al., Nature 433:629-633, 2005.-   Callis et al., Plant Physiol., 88:965-968, 1988.-   Carrington and Freed, J. Virology, 64:1590, 1990.-   Chai et al., Seed Science Research 8 (Supplement 1):23-28, 1998.-   Chandler et al., Plant Cell, 1:1175-1183, 1989-   Chu et al., Sci. Sinica 18:659-668, 1975.-   Chu et al., Scientia Sinica, 18:659-668, 1975.-   Coruzzi et al., EMBO J., 3:1671-1679, 1984.-   Daley et al., Plant Cell Reports 17:489-496 1998.-   Dekeyser et al., Pl. Physiol., 90:217-223, 1989.-   Della-Cioppa et al., Bio/Technology, 5 579-584, 1987.-   Dellaporta et al., In: Chromosome Structure and Function: Impact of    New Concepts, 18th Stadler Genetics Symposium, 11:263-282, 1988.-   Depicker, et al., J. Mol. Appl. Genet. 1: 561-574. 1982.-   Duncan et al., Planta 165:322-332, 1985.s-   Elliot et al., Plant cell Rep., 18:707-714, 1999.-   EP 0385 962-   EP 275,957-   Fraley et al., Proc. Natl. Acad. Sci. USA, 80:4803-4807, 1983.-   Gamborg et al., Exp Cell Res. 50:151-8, 1968.-   Ikatu et al., Bio/Technol., 8:241-242, 1990.-   Jefferson et al., Biochem. Soc. Trans., 15:7-19, 1987a.-   Jefferson et al., EMBO J., 6:3901-3907, 1987b.-   Katz et al., J. Gen. Microbiol., 129:2703-2714, 1983.-   Keller et al., Transgenic Res. 6:385-392, 1997.-   Klee et al., Mol. Gen. Genet., 210:437-442, 1987.-   Komari et al., Plant J. 10: 165-174, 1996.-   Kuhlemeier et al., Plant Cell, 1:471-478, 1989.-   Lawton et al., Plant Mol. Biol. 9:315-324, 1987.-   Linsmaier and Skoog, Physiol. Plant. 18: 100-127, 1965.-   Linsmaier and Skoog, Physiol. Plant., 18 100, 1965.-   Lloyd and McCown, Proc.-Int. Plant Propagator's Soc., 30: 421-427,    1981-   Marcotte et al., Plant Cell, 1:969-976, 1989.-   McCabe & Martinell, Bio/Technology 11:596-598, 1993.-   Miki and McHugh, J. Biotechnol., 107:193-232, 2004.-   Miki et al., In: Methods in Plant Molecular Biology and    Biotechnology, Glick and Thompson ((Eds.), CRC Press, Inc., Boca    Raton, pages 67-88, 1993.-   Murashige and Skoog, Physiol. Plant. 15: 473-497, 1962.-   Nitsch and Nitsch, Science 163:85-87 1969.-   Odell et al., Nature 313:810-812, 1985.-   Oreifig et al., Pl. Cell. Rep. 22:490-496, 2004.-   Ow et al., Science, 234:856-859, 1986.-   PCT Appln. WO 04009761-   PCT Appln. WO 04074443-   PCT Appln. WO 05003362-   PCT Appln. WO 8704181A-   PCT Appln. WO8900193A-   PCT Appln. WO 00/18939-   PCT Appln. WO9215675-   PCT Appln. WO9215775-   PCT Appln. WO9927116-   Sandvang, Antimicrob. Agents Chemotherapy 43:3036-3038, 1999.-   Senaratna et al., Pl. Physiol. 72:620-624, 1983.-   Schaffner et al., Plant Cell, 3:997-1012, 1991-   Schenk and Hildebrandt, Can. J. Bot. 50:199-204, 1972.-   Sutcliffe et al., Proc. Natl. Acad. Sci. USA, 75:3737-3741, 1978.-   Svab et al., Plant Mol. Biol. 14:197-205, 1990.-   Tegeder et al. Pl. Cell Rep. 15:164-169, 1995.-   Uchimiya and Murashige, Plant Physiol. 15:73, 1962.-   Uchimiya and Murashige, Plant Physiol. 57: 424-429, 1976.-   Vertucci and Roos, Pl. Physiol. 90:1019-1023, 1990.-   Walker et al., Proc. Natl. Acad. Sci. USA, 84:6624, 1987-   Wuni et al., Plant Cell, 1:961-968, 1989.-   Yang et al. Proc. Natl. Acad. Sci. USA, 87:4144-4148, 1990.-   Zambre et al., Planta 216:580-586, 2003.-   Zukowsky et al., Proc. Natl. Acad. Sci. USA, 80:1101-1105, 1983.

1. A method for producing a transgenic plant containing at least twoheterologous nucleic acid sequences, comprising: (a) providing anexplant comprising a first heterologous nucleic acid sequence thatconfers resistance to an herbicide, an aminoglycoside, or hygromycin;(b) inoculating the explant with Agrobacterium comprising a vector whichcomprises a second heterologous nucleic acid sequence comprising aselectable marker gene conferring spectinomycin resistance; (c)co-cultivating the explant with the Agrobacterium to obtain an explantthat exhibits spectinomycin resistance; and (d) regenerating the explantthat exhibits spectinomycin resistance into a transgenic plantcontaining at least the two heterologous nucleic acid sequences; whereinthe explant is treated with a cytokinin at least during steps (b) and(c).
 2. The method of claim 1, wherein the explant comprises anembryonic meristem.
 3. The method of claim 1, wherein the firstheterologous nucleic acid sequence confers resistance to glyphosate,bialaphos, phosphinothricin, Basta, glufosinate, 2,4-D, kanamycin, anacetyl-coA carboxylase inhibitor, an oxygen radical generator, ordicamba.
 4. The method of claim 1, wherein the explant is a soybean,corn, cotton, or canola explant.
 5. The method of claim 1, wherein theexplant is a soybean or cotton explant.
 6. The method of claim 1,wherein the transgenic plant arises from transformation of a meristemthat results in transformation of germline tissue.
 7. The method ofclaim 1, wherein the resulting plant is non-chimeric.
 8. The method ofclaim 1, wherein the resulting plant is chimeric.
 9. The method of claim1, wherein the cytokinin is selected from the group consisting ofthidiazuron, BAP (6-Benzylaminopurine), kinetin, CPPU(N-(2-Chloro-4-pyridyl)-N′-phenylurea), 2iP (6-(y,y-Dimethylallylamino)purine), Zeatin, Zeatin-riboside, Adenine, and TIBA(2,3,5-Triiodobenzoic acid).
 10. The method of claim 1, furthercomprising treatment of the explant with a cytokinin prior to step (b).